Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.     

Perceived stress management skill mediates the relationship between optimism and positive mood following radical prostatectomy.

This study evaluated relations among optimism, perceived stress management skills (PSMS), and positive mood in 46 men who had surgical treatment for localized prostate cancer. The authors found that optimism, PSMS, and positive mood scores were positively correlated. Positive mood was unrelated to demographic and disease-related control variables. In a hierarchical regression model controlling for PSMS, the relationship between optimism and positive mood became nonsignificant, whereas PSMS remained a correlate of positive mood. Results suggest that the relationship between optimism and positive mood may be mediated by belief in being able to use stress management techniques effectively. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

THE OPTIMUM SPEED OF VISUAL INSPECTION USING A RANDOM SEARCH STRATEGY

Choosing a standard speed for visual inspection is difficult using standard work measurement techniques. This paper uses a different approach, taking a model of human functioning in inspection and deriving an optimum speed of working so as to balance the cost of time against the cost of errors. Random search is used as the model and both self-paced and externally-paced conditions are derived for multiple fault types. Optimization using a Golden Section search is used to provide optimum inspection speed.     

Defensiveness, trait anxiety, and Epstein-Barr viral capsid antigen antibody titers in healthy college students.

Investigated the relationship of individual differences in repressive coping styles with differences in antibody titer to Epstein-Barr (EB) viral capsid antigen in a normal, healthy college population made up of people previously exposed to EB. Each of 54 1st-yr undergraduates completed a battery of physical-status questions and items pertaining to potential behavioral immunomodulatory confounds, along with the Taylor Manifest Anxiety Scale and the Marlowe-Crowne Social Desirability Scale. Ss reporting high and middle levels of anxiety had higher antibody titers to EB, suggesting poorer immune control over the latent virus, as compared with the low-anxious group. Similarly, high-defensive Ss had higher antibody titers than their low-defensive counterparts, and neither group differed from the middle group. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Kalman-filter-based algorithms of spectrometric datacorrection-Part I: an iterative algorithm of deconvolution

This series of two papers aims to present the different solutions of the problem of improving the resolution of spectrometric measurements via numerical processing of spectrometric data subject both to systematic instrumental errors and to random measurement errors. It is assumed that the model of the spectrometric data has the form of a convolution-type equation of the first kind. The method for improving the resolution consists in numerically solving this equation using the acquired data. In this first paper of the series, an algorithm of correction is proposed which is based on the iterative use of the Kalman filter incorporating a non-negativity constraint. Its applicability to the problem of correction is assessed not only from a purely metrological point of view (accuracy, resolution) but also with respect to its suitability for implementation as a VLSI processor dedicated to measuring systems. For this latter reason a time-invariant model of the data and a steady-state version of the Kalman filter is used. The efficiency of this approach to correction is demonstrated using both synthetic and real-world data     

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.     

Contribution of a disulfide bridge to the stability of 1,3-1,4-P-D-glucan 4-glucanohydrolase from Bacillus licheniformis

Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61A–C90A have 10% of wt V_max. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61A–C90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations     

High-pressure catalytic and thermal cracking of polyethylene

The thermal cracking and catalytic cracking processes of low-density polyethylene were studied in a closed autoclave. The compositions of gaseous and liquid products were analysed by means of GC/FID and GS/MS chromatographic methods. The fractional composition of liquid products was found by distillation. Increased temperature of PE depolymerisation process increases the production of gaseous products and low-boiling liquid compounds; more aromatic hydrocarbons are formed instead of alkenes. When a lower temperature and longer time are adopted for the process to reach the assumed conversion, more straight chained hydrocarbons are produced. The acidic aluminosilicate catalyst yields more low-boiling liquid fractions, more isoalkanes and more aromatics. The neutral alumina is favourable for the production of alkenes and vacuum gas oil fraction in comparison to a non-catalytic process. The Ni–Mo/Al₂O₃ catalyst is efficient in hydrogenation of depolymerisation products. The reaction products contain only saturated compounds then and no aromatics are formed.