Enhanced enzymatic hydrolysis of hydrothermally pretreated empty fruit bunches at high solids loadings by the synergism of hemicellulase and polyethylene glycol

In enzymatic hydrolysis, high lignocellulose loadings are required to obtain high sugar titers. However, the high solids loadings limit enzymatic hydrolysis. In this study, to overcome this limitation, the promoting and synergistic effects of the accessory agents of hemicellulase (i.e., Cellic HTec2) and polyethylene glycol (PEG) 8000 were investigated in the enzymatic hydrolysis of hydrothermally pretreated empty fruit bunches (EFBs). After the optimal addition of Cellic HTec2 and PEG, high enzymatic digestion of the pretreated EFBs was achieved owing to their synergistic effects, even at high solids loadings. For example, the enzymatic digestibility of pretreated EFBs at a 21.7% (w/v) solids loading with 10 FPU of Cellic CTec2/g glucan reached 72.5% when 2.7 mg of Cellic HTec2/g glucan and 62.5 mg of PEG/g glucan were used as the accessory agents. These results suggested that the optimal addition of accessory agents is effective for the enhanced hydrolysis of lignocellulose using even a commercial cellulase preparation.     

Long-Term GABA Treatment Elicits Supersensitivity of Quisqualate-Preferring Metabotropic Glutamate Receptor in Cultured Rat Cerebellar Neurons

Abstract: In primary cultures of rat cerebellar granule neurons, GABA treatment (50 μ M , 7 days) caused a withdrawal supersensitivity selective for the metabotropic glutamate receptors that mainly prefer l -glutamate, quisqua- late and, to a lesser extent, kainate. The withdrawal supersensitivity was absent when 10 μ M SR-95531 was coadministered with GABA during the treatment period, an event that suggests the GABA_A receptors primarily produced the GABA treatment effect. This was supported further by the inability of baclofen treatment to mimic completely the treatment effect of GABA. Withdrawal from 7 days of baclofen treatment only produced a slight increase in the metabotropic effect of l -glutamate and carbachol. In addition, in untreated neurons, baclofen had no acute effect, whereas GABA inhibited the effect of l -glutamate and carbachol. The inhibitory effect of GABA was reversed by SR-95531 and was absent in neurons treated with GABA. These observations suggest the involvement of GABA_A receptors and the apparent development of tolerance to GABA, respectively. Also, dependence on GABA may have occurred; the metabotropic effects of glutamate, kainate, and quisqualate were not altered in neurons maintained with GABA treatment.     

Recent Advances in the use of Immobilized Lipases Directed Toward the Asymmetric Syntheses of Complex Molecules

Water-insoluble compounds can be substrates for enzymatic reactions when lipases are immobilized properly and suitable organic solvents are used. In this review, three type of lipase immobilization method and their application to the asymmetric syntheses of complex molecules are described. Lipases immobilized with Celite or synthetic prepolymers such as urethane prepolymer and photo-crosslinkable resin prepolymer have been applied for the kinetic resolution of many kinds of water-insoluble substrate.

Phospholipid-lipase aggregates with ether linkages are novel and have been found to function effectively as immobilized lipases in asymmetric hydrolysis or esterification reactions in water-saturated organic solvent. The phospholipid-lipase aggregates are considered to have a stacked bilayer based on X-ray diffraction analysis structure of the lipid in the crystalline phase.     

Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC₅₀ values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a K_i value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.     

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic ¹²⁵I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [¹²⁵I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.     

NAD hydrolysis: Chemical and enzymatic mechanisms

The pyridine nucleotide have important non-redox activities as cellular effectors and metabolic regulators [1–3]. The enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond of NAD⁺ and the attendant delivery of the ADPRibosyl moiety to acceptors is central to these many diverse biological activities. Included are the medically important NAD-dependent toxins associated with cholera, diphtheria, pertussis, and related disease [4]; the reversible ADPRibosylation-mediated biological regulatory systems [5,6]; the synthesis of poly (ADPRibose) in response to DNA damage or cellular, division [7]; and the synthesis of cyclic ADPRibose as part of an independent, calcium-mediated regulatory system[8]. As will be presented in this chapter, all evidence points to both the chemical and enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond being dissociative in character via an oxocarbenium intermediate.     

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as ‘complementary’ stress-targets

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25–150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.     

Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED

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Selectivity is an Important Characteristic of Lipases (Acylglycerol Hydrolases)

Lipases are enzymes that usually hydrolyze acylglycerols, but will hydrolyze the carboxylic esters in many other compounds. They also catalyze esteriftcations and transesterifications. In addition to specificity for carboxylic esters, the lipases are selective for lipid classes and show selectivity for primary vs. secondary alcohols (positional or regio-), fatty acids, enantiomers (chirality of either the acid or alcohol residue) and combinations of these. Uses of the enzymes have depended to some extent on regio- and fatty acid selectivities. Newer applications, such as ester synthesis and asymmetric hydrolysis, may not be based on selectivities. Factors affecting selectivities are discussed and some areas for research are mentioned.