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Investigates the possible intermediates in the mechanism of dihydrogen activation by the nickel-iron center of nickel-iron hydrogenases using quantum chemical methods. Structural and electronic properties of the active site models; Spectroscopical identification of the enzymes; Redox chemistry of the hydrogenases; Role of the cysteine ligands in the reactions.

The multi-domain protein hSos1 plays a major role in cell growth and differentiation through its Ras-specific guanine nucleotide exchange domain whose complex regulation involves intra-molecular, inter-domain rearrangements. We present a stochastic mathematical model describing intra-molecular regulation of hSos1 activity. The population macroscopic effect is reproduced through a Monte-Carlo approach. Key model parameters have been experimentally determined by BIAcore analysis. Complementation experiments of a Saccharomyces cerevisiae cdc25(ts) strain with Sos deletion mutants provided a comprehensive data set for estimation of unknown parameters and model validation. The model is robust against parameter alteration and describes both the behavior of Sos deletion mutants and modulation of activity of the full length molecule under physiological conditions. By incorporating the calculated effect of amino acid changes at an inter-domain interface, the behavior of a mutant correlating with a developmental syndrome could be simulated, further validating the model. The activation state of Ras-specific guanine nucleotide exchange domain of hSos1 arises as an "emergent property" of its multi-domain structure that allows multi-level integration of a complex network of intra- and inter-molecular signals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chain-length preference profile, increasing the relative activity towards C8 substrates. Moreover, mutations conferred to PFL higher temperature stability. On the other hand, replacement of the serine at position 141 by glycine destabilized the protein.

Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or LipMRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (LipMRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation. [ABSTRACT FROM AUTHOR]

Objectives: The lipase gene lipSR1 isolated from oil-contaminated soil exhibits high hydrolytic activity for short-chain fatty acid substrates. A single calcium ion is required to anchor the lid of LipSR1 in an open conformation by coordination with two aspartate residues and three other residues in the lid. The lid of LipSR1 is anchored by Ca2+, which is coordinated by side-chain carboxyl oxygens of Asp153 and Asp157, carbonyl oxygens of Thr118 and Ser144, and the side chain of Gln120. Results: D157A, D153R, Q120A, S144A, and T118A mutants were produced by site-directed mutagenesis in this study. Analyses of hydrolytic activity and thermostability showed that the properties of D157A, D153R, Q120A, and S144A were almost lost, suggesting that Asp157, Asp153, Gln120, and Ser144 are important residues for LipSR1. However, the catalytic performance of T118A was clearly maintained. Moreover, the thermostability of mutant T118A was higher than that of wild-type LipSR1. Conclusions: These results indicated that mutation of threonine at position 118 improved the stability of the enzyme at high temperature. [ABSTRACT FROM AUTHOR]

Proper mitochondrial performance is imperative for the maintenance of normal neuronal function to prevent the development of neurodegenerative diseases. Persistent accumulation of damaged mitochondria plays a role in prion disease pathogenesis, which involves a chain of events that culminate in the generation of reactive oxygen species and neuronal death. Our previous studies have demonstrated that PINK1/Parkin-mediated mitophagy induced by PrP106-126 is defective and leads to an accumulation of damaged mitochondria after PrP106-126 treatment. Externalized cardiolipin (CL), a mitochondria-specific phospholipid, has been reported to play a role in mitophagy by directly interacting with LC3II at the outer mitochondrial membrane. The involvement of CL externalization in PrP106-126-induced mitophagy and its significance in other physiological processes of N2a cells treated with PrP106-126 remain unknown. We demonstrate that the PrP106-126 peptide caused a temporal course of mitophagy in N2a cells, which gradually increased and subsequently decreased. A similar trend in CL externalization to the mitochondrial surface was seen, resulting in a gradual decrease in CL content at the cellular level. Inhibition of CL externalization by knockdown of CL synthase, responsible for de novo synthesis of CL, or phospholipid scramblase-3 and NDPK-D, responsible for CL translocation to the mitochondrial surface, significantly decreased PrP106-126-induced mitophagy in N2a cells. Meanwhile, the inhibition of CL redistribution significantly decreased PINK1 and DRP1 recruitment in PrP106-126 treatment but had no significant decrease in Parkin recruitment. Furthermore, the inhibition of CL externalization resulted in impaired oxidative phosphorylation and severe oxidative stress, which led to mitochondrial dysfunction. Our results indicate that CL externalization induced by PrP106-126 on N2a cells plays a positive role in the initiation of mitophagy, leading to the stabilization of mitochondrial function. [ABSTRACT FROM AUTHOR]

In traditional cheese making, pregastric lipolytic enzymes of animal origin are used for the acceleration of ripening and the formation of spicy flavor compounds. Especially for cheese specialities, such as Pecorino, Provolone, or Feta, pregastric esterases (PGE) play an important role. A lipase from Pleurotus citrinopileatus could serve as a substitute for these animal-derived enzymes, thus offering vegetarian, kosher, and halal alternatives. However, the hydrolytic activity of this enzyme towards long-chain fatty acids is slightly too high, which may lead to off-flavors during long-term ripening. Therefore, an optimization via protein engineering (PE) was performed by changing the specificity towards medium-chain fatty acids. With a semi-rational design, possible mutants at eight different positions were created and analyzed in silico. Heterologous expression was performed for 24 predicted mutants, of which 18 caused a change in the hydrolysis profile. Three mutants (F91L, L302G, and L305A) were used in application tests to produce Feta-type brine cheese. The sensory analyses showed promising results for cheeses prepared with the L305A mutant, and SPME-GC-MS analysis of volatile free fatty acids supported these findings. Therefore, altering the chain length specificity via PE becomes a powerful tool for the replacement of PGEs in cheese making. [ABSTRACT FROM AUTHOR]

Cold active esterases have gained great interest in several industries. The recently determined structure of a family IV cold active esterase (EstN7) from Bacillus cohnii strain N1 was used to expand its substrate range and to probe its commercially valuable substrates. Database mining suggested that triacetin was a potential commercially valuable substrate for EstN7, which was subsequently proved experimentally with the final product being a single isomeric product, 1,2-glyceryl diacetate. Enzyme kinetics revealed that EstN7's activity is restricted to C2 and C4 substrates due to a plug at the end of the acyl binding pocket that blocks access to a buried water-filled cavity. Residues M187, N211 and W206 were identified as key plug forming residues. N211A stabilised EstN7 allowing incorporation of the destabilising M187A mutation. The M187A-N211A double mutant had the broadest substrate range, capable of hydrolysing a C8 substrate. W206A did not appear to have any significant effect on substrate range either alone or when combined with the double mutant. Thus, the enzyme kinetics and engineering together with a recently determined structure of EstN7 provide new insights into substrate specificity and the role of acyl binding pocket plug residues in determining family IV esterase stability and substrate range. [ABSTRACT FROM AUTHOR]

The authors discuss a study which analyzed a large database of nonsmokers from different geographical areas of China to report on the association of household air pollution (HAP) and secondhand cigarette smoke (SHS) with lung cancer mortality. Topics include increase in lung cancer-related death for every five years of follow-up, proportions of the age-standardized mortality rate of lung cancer attributable to HAP from solid fuels, and the lack of association between SHS and lung cancer.

Aspergillus oryzae lipase (AOL) is a potential biocatalyst for industrial application. In this study, a mutant lipase AOL-3F38N/V230R was screened through two rounds of directed evolution, resulting in a fourfold increase in lipase activity, and threefold in catalytic efficiency (kcat/Km), while maintaining its excellent stereoselectivity. AOL-3F38N/V230R enzyme activity was maximum at pH 7.5 and also at 40 °C. And compared with wild-type AOL-3, AOL-3F38N/V230R preferentially hydrolyzed the fatty acid ethyl ester carbon chain length from C4 to C6–C10. In the same catalytic reaction conditions, the conversion of (R,S)-ethyl-2-(4-hydroxyphenoxy) propanoate ((R,S)-EHPP) by AOL-3F38N/V230R can be increased 169.7% compared to the original enzyme. The e.e.s of (R,S)-EHPP achieved 99.4% and conversion about 50.2% with E value being 829.0. Therefore, AOL-3F38N/V230R was a potential biocatalyst for obtaining key chiral compounds for aryloxyphenoxy propionate (APP) herbicides. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a common zoonotic bacterium for disease in pigs and human and causes health problem. The aim of the study is to investigate the potential of soluble pathogenic recognition protein pentraxin-3 (PTX3) on inhibiting proliferation, adhesion, and infection of S. aureus in porcine kidney epithelial PK15 cells (PK15 cells). PTX3 suppressed the growth of S. aureus and avoided it adhering to PK15 cells. PTX3 treatment also attenuated cell apoptosis induced by S. aureus in PK15 cells. The results suggest that PTX3 is able to interfere with the bacterial pathogenesis via inhibiting the growth of S. aureus and decreasing their adhesion to cells. Hence, PTX3 may replace the use of antibiotics and develop as a novel anti-microbial agent for disease prevention in piglets. [ABSTRACT FROM AUTHOR]

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts. [ABSTRACT FROM AUTHOR]

The authors discuss a study published within the issue which present a novel molecular link among pentraxin (PTX) 3 deficiency, amplified complement activation, and granuloma formation in experimental models and in patients with sarcoidosis. Topics include clinical characteristics of sarcoidosis, the proneness of PTX3-deficient mice to exacerbation of the myocbacterium superoxide, and the work's shortcomings that provide ample opportunity for future study.

CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency ( k / K ) toward palmitic acid compared to wild-type, respectively. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported. [ABSTRACT FROM AUTHOR]

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T1 lipase is a potential biocatalyst for industrial application since it is highly thermostable and displays optimal activity at 60-75°C. Structural analysis of T1 lipase shows that its lid region undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation. To study structure/function of this atypical lid, we performed site-directed mutagenesis on the hydrophobic residues in the lid region. These residues were mutated to hydrophilic ones and biochemical properties of mutants were investigated. Results showed that F181 might be an important residue for enzyme-substrate binding. Mutants A186S and A190S had 35-50% increase in catalytic efficiencies compared to wild-type T1, without compromising their functions at high temperatures. In general, mutagenesis did not cause large changes to chain-length preference in T1 lipase. Mutants A186S and V187N were inactive towards long-chain pNP esters (p-Nitrophenyl stearate) and V187N showed lower activity towards long-chain triacylglycerols than wt T1, which makes it a potential catalyst in dairy industry. Thermostability of mutants were affected at different extent due to the influence of hydrophobic contact between the lid and the protein core. These findings not only shed light on the lipase structure/function relationship but also lay the framework for further engineering to gain more potent, stable, and selective lipases. Practical applications: A thermostable T1 lipase owns an atypical lid and hydrophobic residues in the lid influence its catalytic properties. Here, we screened mutants A186S and A190S with 35-50% increase in catalytic efficiency, which have industrial application in the modification of fats and oils. Moreover, mutant V187N showed lower activity towards long-chain TAGs than wt T1. This mutant can be a potential biocatalyst in dairy industry which promote generation of short-chain fatty acids from milk fats to increase flavors in dairy products. The lid region of thermostable T1 lipase undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation (PDB ID: 2DSN, 2W22 [16,17]). Mutagenesis on hydrophobic residues in the lid affect catalytic properties of T1 lipase. [ABSTRACT FROM AUTHOR]

Enzymatic activity is important characteristic of enzymes for industrial application, which can be improved by protein engineering. PLA1 is a biocatalyst applied in phospholipid modification for its phospholipase activity and had obtained board attention for its application in oil degumming. Bioinformatics analysis suggested that three charged residues, R81, R84, and E87 located in the lid of the protein, may affect the conformational change of lid thus influence the activity of the enzyme. In the current study, mutagenesis of these residues was conducted with protein engineering. Five mutants such as R81A, R84A, R84M, R84K, and E87Q with higher phospholipase activity were screened out. Biochemical properties analysis showed that all of them had identical optimal pH value with wild-type, while the optimal temperature was decreased to be 50°C and the kcat /KM was improved. Degumming soybean oil, three of five mutants, R81A, R84M, and E87Q, decreased phosphorous content lower than 8.3 mg/kg within 3 h, which was highly improved compared with wild-type. R84M decrease phosphorus content less than 5 mg/kg within 5 h. These findings not only permit optimization of enzyme performance in degumming but also shed light on the application of bioinformatics techniques and protein engineering techniques on industry application. Practical applications: The results support that design proteins according to bioinformatics and protein engineering is desirable and the phospholipase with higher activity is more suitable for oil degumming. The phospholipase activity of PLA1 can be improved by protein engineering based on bioinformatics analysis. With its properties of hydrolyzing sn-1 position ester bond of phospholipids, nonhydratable phospholipids were converted into their hydratable forms, PLA1 can be applied in oil degumming. PLA1 with higher phospholipase activity is more likely to suitable for oil degumming since it could decrease the phosphorous content of oil lower than 10 mg/kg within shorter reaction, which showed great potencial for degumming application. [ABSTRACT FROM AUTHOR]

Cold active lipases (CLPs) are gaining importance nowadays as they are increasingly used in fine chemical synthesis, bioremediation, food processing and as detergent additive. These enzymes exhibit high catalytic activity at low temperatures and flexibility to act at low water medium. Since they are active at low temperatures consume less energy and also stabilize fragile compounds in the reaction medium. CLPs are commonly obtained from psychrophilic microorganisms which thrive in cold habitats. Compared to mesophilic and thermophilic lipases, only a few CLPs were studied and industrially exploited so far. CLPs (C. antarctica lipase-A and C. antarctica lipase-B) from Candida antarctica isolated from Antarctic region are the well studied and industrially employed, and many are being followed up. This review updates the CLPs reported recently and the industrial applications of CLPs. [ABSTRACT FROM AUTHOR]

The acute phase protein Pentraxin 3 (PTX3) plays a non-redundant role as a soluble pattern recognition receptor for selected pathogens and it represents a rapid biomarker for primary local activation of innate immunity and inflammation. Recent evidence indicates that PTX3 exerts an important role in modulating the cardiovascular system in humans and experimental models. In particular, there are conflicting points concerning the effects of PTX3 in cardiovascular diseases (CVD) since several observations indicate a cardiovascular protective effect of PTX3 while others speculate that the increased plasma levels of PTX3 in subjects with CVD correlate with disease severity and with poor prognosis in elderly patients. In the present review, we discuss the multifaceted effects of PTX3 on the cardiovascular system focusing on its involvement in atherosclerosis, endothelial function, hypertension, myocardial infarction and angiogenesis. This may help to explain how the specific modulation of PTX3 such as the use of different dosing, time, and target organs could help to contain different vascular diseases. These opposite actions of PTX3 will be emphasized concerning the modulation of cardiovascular system where potential therapeutic implications of PTX3 in humans are discussed. [ABSTRACT FROM AUTHOR]

Metabolic pathways of aerobic bacteria able to assimilate sulfur can provide biocatalysts for biodesulfurization of petroleum and of other sulfur-containing pollutants. Of major interest is the so-called '4S pathway,' in that C-S bonds are specifically cleaved leaving the carbon skeleton of substrates intact. This pathway is carried out by four enzymes, named Dsz A, B, C, and D. In view of a possible application of recombinant Dsz enzymes in biodesulfurization treatments, we have investigated the structural features of enzymes cloned from a Rhodococcus strain isolated from polluted environmental samples and their resistance to temperature (20-95 °C) and to organic solvents (5, 10, and 20 % v/v methanol, acetonitrile, hexane, and toluene). Changes in protein structures were assessed by circular dichroism and intrinsic fluorescence spectroscopy. We found that all Dsz proteins are unfolded by temperatures in the range 45-60 °C and by all solvents tested, with the most dramatic effect being produced by toluene. These results suggest that stabilization of the biocatalysts by protein engineering will be necessary for developing biodesulfurization technologies based on Dsz enzymes. [ABSTRACT FROM AUTHOR]

A low-temperature-active alkaline esterase, Est12, from a marine sediment metagenomic fosmid library was identified. Est12 prefers short- and middle-chain p-nitrophenol esters as substrate with optimum temperature and pH value of 50 °C and 9.0, respectively, and nearly 50 % of maximum activity retained at 5 °C. The hydrolysis activity of Est12 was stable at 40 °C. Ca especially activated the activity of Est12 to about 151 % of the control. DEPC and PMSF inhibited the activity of Est12 to 34 and 25 %, respectively. In addition, Est12 was more tolerable to methanol compared to other organic solvents tested. The crystal structure of Est12 at 1.39 Å resolution showed that the cap domain which is composed of an α-helix and a flexible region resulted in a relatively wide spectrum of substrate, with p-nitrophenol caproate as the preferred one. Furthermore, the flexible cap domain and the high percentage of Gly, Ser, and Met may play important roles in the adaptation of Est12 to low temperature. [ABSTRACT FROM AUTHOR]

Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. [ABSTRACT FROM AUTHOR]

The fragment 106-126 of prion protein exhibits similar properties to full-length prion. Experiments have shown that the A117V mutation enhances the aggregation of PrP106-126, while the H111S mutation abolishes the assembly. However, the mechanism of the change in the aggregation behavior of PrP106-126 upon the two mutations is not fully understood. In this study, replica exchange molecular dynamics simulations were performed to investigate the conformational ensemble of the WT PrP106-126 and its two mutants A117V and H111S. The obtained results indicate that the three species are all intrinsically disordered but they have distinct morphological differences. The A117V mutant has a higher propensity to form β-hairpin structures than the WT, while the H111S mutant has a higher population of helical structures. Furthermore, the A117V mutation increases the hydrophobic solvent accessible surface areas of PrP106-126 and the H111S mutation reduces the exposure of hydrophobic residues. It can be concluded that the difference in populations of β-hairpin structures and the change of hydrophobic solvent accessible areas may induce the different aggregation behaviors of the A117V and the H111S mutated PrP106-126. Understanding why the two mutations have contrary effects on the aggregation of PrP106-126 is very meaningful for further elucidation of the mechanism underlying aggregation and design of inhibitor against aggregation process. [ABSTRACT FROM AUTHOR]

ABSTRACT In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein, and, in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years because of implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with buried active sites and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. Proteins 2015; 83:599-611. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considereda potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273-278 and the 3104 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6℃ and 187 min, respectively, while that was 54.6℃ and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications. [ABSTRACT FROM AUTHOR]

Several Pseudomonas sp. CR611 Lip I.3 mutants with overall increased activity and a shift towards longer chain substrates were constructed. Substitution of residues Y29 and W310 by smaller amino acids provided increased activity on C18-substrates. Residues G152 and S154, modified to study their influence on interfacial activation, displayed a five and eleven fold increased activity. [ABSTRACT FROM AUTHOR]

Biodiesel is an environment-friendly and renewable fuel produced by transesterification of various feedstocks. Although the lipase-catalyzed biodiesel production has many advantages over the conventional alkali catalyzed process, its industrial applications have been limited by high-cost and low-stability of lipase enzymes. This review provides a general overview of the recent advances in lipase engineering, including both protein modification and production. Recent advances in biotechnology such as in protein engineering, recombinant methods and metabolic engineering have been employed but are yet to impact lipase engineering for cost-effective production of biodiesel. A summary of the current challenges and perspectives for potential solutions are also provided. Biotechnol. Bioeng. 2014;111: 639-653. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In an era of economic globalization, the competition among wine businesses is likely to get tougher. Biotechnological innovation permeates the entire world and intensifies the severity of the competition of the wine industry. Moreover, modern consumers preferred individualized, tailored, and healthy and top quality wine products. Consequently, these two facts induce large gaps between wine production and wine consumption. Market-orientated yeast strains are presently being selected or developed for enhancing the core competitiveness of wine enterprises. Reasonable biological acidity is critical to warrant a high-quality wine. Many wild-type acidity adjustment yeast strains have been selected all over the world. Moreover, mutation breeding, metabolic engineering, genetic engineering, and protoplast fusion methods are used to construct new acidity adjustment yeast strains to meet the demands of the market. In this paper, strategies and concepts for strain selection or improvement methods were discussed, and many examples based upon selected studies involving acidity adjustment yeast strains were reviewed. Furthermore, the development of acidity adjustment yeast strains with minimized resource inputs, improved fermentation, and enological capabilities for an environmentally friendly production of healthy, top quality wine is presented. [ABSTRACT FROM AUTHOR]

Bovine spongiform encephalopathy (BSE), or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP), which adopts two conformers; PrPC is the native innocuous form, which is a-helix rich; and PrPSc is the β-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative β-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative β-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative β-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative β-strand formation, thereby improving our understanding of how PrPC misfolds into the β-sheet rich PrPSc and how pH factors into the process. [ABSTRACT FROM AUTHOR]

Prion diseases are marked by cerebral accumulation of the abnormal isoform of the prion protein. A fragment of prion protein composed of residues 106–126 (PrP106–126) exhibits similar properties to full length prion and plays a key role in the conformational conversion from cellular prion to its pathogenic pattern. Soluble oligomers of PrP106–126 have been proposed to be responsible for neurotoxicity. However, the monomeric conformational space and initial oligomerization of PrP106–126 are still obscure, which are very important for understanding the conformational conversion of PrP106–126. In this study, replica exchange molecular dynamics simulations were performed to investigate monomeric and dimeric states of PrP106–126 in implicit solvent. The structural diversity of PrP106–126 was observed and this peptide did not acquire stable structure. The dimeric PrP106–126 also displayed structural diversity and hydrophobic interaction drove the dimerization. To further study initial oligomerization of PrP106–126, 1 µs conventional molecular dynamics simulations of trimer and tetramer formation were carried out in implicit solvent. We have observed the spontaneous formation of several basic oligomers and stable oligomers with high β-sheet contents were sampled in the simulations of trimer and tetramer formation. The β-hairpin formed in hydrophobic tail of PrP106–126 with residues 118–120 in turn may stabilize these oligomers and seed the formation oligomers. This study can provide insight into the detailed information about the structure of PrP106–126 and the dynamics of aggregation of monomeric PrP106–126 into oligomers in atomic level. [ABSTRACT FROM AUTHOR]

Aspergillus niger phytase (PhyA) has been used as a feed supplement to improve the bioavailability of phytate phosphorus to swine and poultry. However, it is unable to maintain its stability due to high temperature during the feed pelleting process. In this study, we performed site-directed mutagenesis in the Aspergillus niger N25 phyA gene at residue 44I and 252 T, and they were replaced by glutamic acid and arginine. Single-site mutants I44E-PhyA and T252R-PhyA, as well as double-site mutant I44E/T252R-PhyA, were constructed to improve the thermostability of PhyA through hydrogen bondings and ionic interactions. The three mutant enzymes all showed more than 20 % improvement in thermostability compared to the wild-type enzyme after being heated at 80 °C for 10 min. Their melting temperatures ( T) were increased by 1, 1, and 1.2 °C, respectively. The k values of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA for sodium phytate were 78, 44, and 79 % lower ( P <0.05) than that of the wild-type enzyme. Overall catalytic efficiency ( k/ k) of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA was improved by 310, 155, and 84 % ( P <0.05) than that of the wild type, respectively. The catalytic efficiency did not seem to be negatively affected by the improvement in thermostability. [ABSTRACT FROM AUTHOR]

Systems Biology holds that complex cellular functions are generated as system-level properties endowed with robustness, each involving large networks of molecular determinants, generally identified by "omics" analyses. In this paper we describe four basic cancer cell properties, that can easily be investigated in vitro: enhanced proliferation, evasion from apoptosis, genomic instability, inability to undergo oncogene induced senescence. Focusing our analysis on a K-ras dependent transformation system, we show that enhanced proliferation and evasion from apoptosis are closely linked, and present findings that indicate how a large metabolic remodeling sustains the enhanced growth ability. Network analysis of transcriptional profiling gives the first indication on this remodeling, further supported by biochemical investigations and metabolic flux analysis. Enhanced glycolysis, down regulation of TCA cycle, decoupling of glucose and glutamine utilization, with increased reductive carboxylation of glutamine, so to yield a sustained production of growth building blocks and glutathione, are the hallmarks of enhanced proliferation. Low glucose availability specifically induces cell death in K-ras transformed cells, while PKA activation reverts this effect, possibly through at least two mitochondrial targets. The central role of mitochondria in determining the two investigated cancer cell properties is finally discussed. Taken together the findings reported herein indicate that a system-level property is sustained by a cascade of interconnected biochemical pathways, that behave differently in normal and in transformed cells. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the role of NACHT, LRR and PYD domains-containing protein (NALP)3 inflammasome in the release of interleukin-1(IL-1)beta from lipopolysaccharide (LPS)-primed microglia after exposure to a synthetic neurotoxic prion fragment (PrP106-126). The production of IL-1beta were assessed using enzyme-linked immunosorbent assay (ELISA). The involvement of NALP3 inflammasome in prion peptide-induced microglial activation is highlighted.

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous biocatalysts known to catalyze the hydrolysis of water insoluble triglycerides in aqueous medium and carry out the reverse reaction (synthesis) under organic solvent rich medium. Microbial lipases have received a great deal of attention in the field of food technology, pharmaceutical sciences, chemical and detergent industries due to their stability, selectivity, mild operation conditions and broad substrate specificity. Despite these advantages, low activity and stability displayed in organic medium has restricted their commercial application in organic synthesis. Researchers have explored alternative ways to modify the enzymes making them suitable for use in non-conventional media. In this context, harvesting lipases from 'Solvent Tolerant Microbes' has recently become an attractive approach. These microbes are able to grow in the presence of high concentrations of organic solvents, generally known to have detrimental effect on microorganisms. Such microbes survive through novel adaptation mechanisms and secretion of solvent stable enzymes having efficient functionality in solvent-rich media. These enzymes could be useful for bioconversion in non-conventional media. In the current review, this approach is described with an emphasis on characteristics, applications and genetic aspect of lipases from the genus Pseudomonas. [ABSTRACT FROM AUTHOR]

A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86-34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH4)2SO4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15-35 °C and pH 5-9, with the optimal conditions being 15-25 °C and pH 5-6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold-active lipase. Its activity was found to increase in the presence of Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short- and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction and oxidative stress are central to the molecular basis of several human diseases associated with neuromuscular disabilities. We hypothesize that mitochondrial dysfunction also contributes to the neuromuscular symptoms observed in patients with ethylmalonic aciduria and homozygosity for ACADS c.625G>A-a common variant of the short-chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) enzyme in the mitochondrial fatty acid oxidation pathway. This study sought to identify the specific factors that initiate cell dysfunction in these patients. We investigated fibroblast cultures from 10 patients with neuromuscular disabilities, elevated levels of ethylmalonic acid (EMA) (>50 mmol/mol creatinine), and ACADS c.625G>A homozygosity. Functional analyses, i.e., ACADS gene and protein expression as well as SCAD enzyme activity measurements, were performed together with a global nano liquid chromatography tandem mass spectroscopy (nano-LC-MS/MS)-based screening of the mitochondrial proteome in patient fibroblasts. Moreover, cell viability of patient fibroblasts exposed to menadione-induced oxidative stress was evaluated. Loss of SCAD function was detected in the patient group, most likely due to decreased ACADS gene expression and/or elimination of misfolded SCAD protein. Analysis of the mitochondrial proteome in patient fibroblasts identified a number of differentially expressed protein candidates, including reduced expression of the antioxidant superoxide dismutase 2 (SOD2). Additionally, patient fibroblasts demonstrated significantly higher sensitivity to oxidative stress than control fibroblasts. We propose that reduced mitochondrial antioxidant capacity is a potential risk factor for ACADS c.625G>A-associated ethylmalonic aciduria and that mitochondrial dysfunction contributes to the neurotoxicity observed in patients. [ABSTRACT FROM AUTHOR]

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so-called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position-restrained MD simulations. Our conclusions indicate that the sole mobility of α9 helix side-chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement. The role of selected α5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase. Proteins 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Human prion diseases are characterized by the conversion of the normal host cellular prion protein (PrPC) into an abnormal misfolded form [disease-associated prion protein (PrPSc)]. Antibodies that are capable of distinguishing between PrPC and PrPSc may prove to be useful, not only for the diagnosis of these diseases, but also for a better understanding of the molecular mechanisms involved in disease pathogenesis. In an attempt to produce such antibodies, we immunized mice with an aggregated peptide spanning amino acid residues 106 to 126 of human PrP (PrP106–126). We were able to isolate and single cell clone a hybridoma cell line (P1:1) which secreted an IgM isotype antibody [monoclonal antibody (mAb P1:1)] that recognized the aggregated, but not the monomeric form of the immunogen. When used in immunoprecipitation assays, the antibody did not recognize normal PrPC from non-prion disease brain specimens, but did selectively immunoprecipitate full-length PrPSc from cases of variant and sporadic Creutzfeldt–Jakob disease and Gerstmann–Straussler–Scheinker disease. These results suggest that P1:1 recognizes an epitope formed during the structural rearrangement or aggregation of the PrP that is common to the major PrPSc types found in the most common forms of human prion disease. [ABSTRACT FROM AUTHOR]

Aspergillus niger phytase (PhyA) has been used as a feed supplement to reduce manure phosphorus excretion of swine and poultry but lacks sufficient thermostability for feed pelleting and appropriate pH-activity profile for phytate hydrolysis in the stomach of animals. Previously, a thermostable mutant PhyA18 and two pH-activity profile-improved mutants E228K and K300E were developed. In this study, the mutations were combined to determine if both improvements were cumulative. Four substitutions (S149P, F131L, K112R, and K195R) identified from random mutagenesis were added sequentially to the combined mutants to further improve their thermostability. Mutant E228K shifted the optimum pH of the parent one from 5.5 to 4.0 and increased ( P < 0.05) its specific activity at pH 3.5, whereas mutant K300E eliminated the activity dip at pH 3.5 shown in the wild type. Mutant S149P further improved thermostability over PhyA18. Our results illustrate the feasibility and structural basis to improve thermostability and pH-activity profile of PhyA phytase by assembling mutations derived from rational design and random mutagenesis. [ABSTRACT FROM AUTHOR]