Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

The effect of ionic liquid media on activity,selectivity and stability of Candida antarctica lipase B in transesterification reactions

Nineteen different 1,3-dialkylimidazolium-based ionic liquids (ILs) were used as reaction media for the synthesis of butyl butyrate by transesterification from vinyl butyrate and 1-butanol catalyzed by Candida antarctica lipase B (CaLB). The reaction was also carried out in hexane as a reference solvent. In all the water-immiscible ILs assayed, the enzymatic activity and selectivity were higher than that obtained in hexane. However, in water-miscible ILs, the activity was lower than in the reference solvent, although they showed >99.99% selectivity. Two solvent properties, hydrophobicity and nucleophilicity, were considered key parameters for analyzing the behavior of CaLB in ILs. In the case of ILs based on the same anion, the synthetic activity was gradually enhanced by increasing cation hydrophobicity. Furthermore, the activity of CaLB was greater in ILs containing anions of lower nucleophilicity. Stability studies indicate that CaLB exhibited greater stability in water-immiscible ILs than in water-miscible ILs.     

Optimization of the covalent coupling and ionic adsorption of magnetic nanoparticles on Flavobacterium ATCC 27551 using the Taguchi method

Abstract

Flavobacterium ATCC 27551 was used as a model system for the preparation of magnetic biocatalysts. The magnetic modification was carried out by covalently binding carboxylate- and amino-modified magnetic nanoparticles onto cells. Magnetic Fe₃O₄ nanoparticles were also used for ionic adsorption on the cell surface. Magnetically modified cells were concentrated using a magnet and exhibited organophosphate hydrolyzing activity. The Taguchi method was used to optimize the binding of the magnetic nanoparticles on the cell surface. SEM image analyses demonstrated good linkage of the magnetic nanoparticles over the Flavobacterium ATCC 27551 cell surface. Under optimal conditions, the magnetic cells displayed specific activity ratios of 93%, 89% and 95%, compared with untreated cells, after the covalent coupling with carboxylate- and amino-modified magnetic nanoparticles and the ionic adsorption of magnetic Fe₃O₄ nanoparticles, respectively.     

A Three-Dimensional Musculoskeletal Model of the Human Knee Joint. Part 2: Analysis of Ligament Function

A three-dimensional model of the knee is used to study ligament function during anterior-posterior (a-p) draw, axial rotation, and isometric contractions of the extensor and flexor muscles. The geometry of the model bones is based on cadaver data. The contacting surfaces of the femur and tibia are modeled as deformable; those of the femur and patella are assumed to be rigid. Twelve elastic elements are used to describe the geometry and mechanical properties of the cruciate ligaments, the collateral ligaments, and the posterior capsule. The model is actuated by thirteen musculotendinous units, each unit represented as a three-element muscle in series with tendon. The calculations show that the forces applied during a-p draw are substantially different from those applied by the muscles during activity. Principles of knee-ligament function based on the results of in vitro experiments may therefore be overstated. Knee-ligament forces during straight a-p draw are determined solely by the changing geometry of the ligaments relative to the bones: ACL force decreases with increasing flexion during anterior draw because the angle between the ACL and the tibial plateau decreases as knee flexion increases; PCL force increases with increasing flexion during posterior draw because the angle between the PCL and the tibial plateau increases. The pattern of ligament loading during activity is governed by the geometry of the muscles spanning the knee: the resultant force in the ACL during isometric knee extension is determined mainly by the changing orientation of the patellar tendon relative to the tibia in the sagittal plane; the resultant force in the PCL during isometric knee flexion is dominated by the angle at which the hamstrings meet the tibia in the sagittal plane.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

Enzymatic synthesis of perillyl alcohol derivatives and investigation of their antiproliferative activity

The monoterpene perillyl alcohol (POH), an intermediate in the plant terpenoid biosynthetic pathway, has well-established tumor chemopreventive and chemotherapeutic potential. We have previously shown that the primary hydroxyl group of POH is essential for its antitumor and anti-angiogenic activities. In the current study we present the enzymatic synthesis of two POH derivatives with different polar and hydrophobic characteristics, namely perillyl glucoside and perillyl glucoside fatty ester, through a two-step modification. Initial glucosylation of POH on its active hydroxyl group with D-(+)-glucose and subsequent esterification of the perillyl glucoside product with vinyl laurate were carried out using almond β-glucosidase and lipase B from Candida antarctica, respectively, in a low-water system. Optimization of enzymatic reactions was performed to achieve the highest possible conversion yields. The antitumor cell proliferation activity against mouse Lewis lung carcinoma cells was retained in both derivatives, although the perillyl glucoside ester showed greater inhibition than perillyl glucoside. Our results underline the feasibility of enzymatically producing novel bioactive analogs of phytochemicals displaying useful physicochemical properties.     

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO₂ (SC-CO₂). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO₂ exposure by up to 15%. L-AAO was more stable in supercritical CO₂ than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO₂, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO₂. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3?h per change of enzyme activity after 3?h was the highest in SC-CO₂ (1.474?mmol?U^?1), where catalase was present, and the lowest in the EMR (0.457?mmol?U^?1).     

Commercial lipase immobilization on Accurel MP 1004 porous polypropylene

Three commercial lipases (CLs), A Amano 6 (from Aspergillus niger), M Amano 10 (from Mucor javanicus), and R Amano (from Penicillium roqueforti) – called lipase A, M and R respectively – were characterized in terms of carbohydrate content, protein content and enzymatic activity (p-nitrophenylacetate assay). All the CL preparations contained different proteins as observed from electrophoresis. Lipases were immobilized on Accurel MP1004 porous polypropylene by physical adsorption.The Immobilization process caused a loss of enzymatic activity. The retained activity was similar for lipase M and R (about 15%). In contrast, lipase A retained only the 1.3% of the specific activity of the free lipase. The retained activity of lipases M and R seems to be due to a feature of the support, while the lower activity a of lipase A may be attributed to a strong structure distortion caused by lipase–support interaction.     

Standardization in Biological Staining. The Influence of Dye Manufacturing

The purpose of biological staining is to obtain specimens of biological material that can be assessed in the microscope. These specimens are influenced by all processes from removal from the intact organism to mounting on the microscopic slide. To achieve comparable results with various techniques for biological staining, standardization of all procedures and reagents is mandatory. In this paper, I focus particularly on dyes and consider the possibilities for obtaining standardized dyes. In general practice, most biological staining takes place with available commercial dyes. These dyes may or may not have been subjected to quality assessment either internally by the producer or vendor or externally by independent investigators or organizations such as the Biological Stain Commission. Concerted attempts at standardization in Europe are discussed. The latest results of this work, the European standard EN 12376, is presented. This standard is concerned with information supplied by the manufacturer with in vitro diagnostic reagents for biological staining. The standard has been prepared by a Working Group on Staining in Biology under Technical Committee 140, In Vitro Medical Devices, of the European committee for standardization, CEN.