Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy

The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using a water-soluble spin relaxation agent, chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (<0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.     

Effect of the lipid interface on the catalytic activity and spectroscopic properties of a fungal lipase

Lipase from the fungi Thermomyces (formerly Humicola) lanuginosa (TlL) is widely used in industry. This interfacial enzyme is inactive under aqueous conditions, but catalytic activation is induced on binding to a lipid-water interface. In order for protein engineering to design more efficient mutants of TlL for specific applications, it is important to characterize its interfacial catalysis. A complete analysis of steady-state kinetics for the hydrolysis of a soluble substrate by TlL has been developed using an interface different from the substrate. Small vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or other anionic phospholipids are a neutral diluent interface for the partitioning of substrate and enzyme. TlL binds to these interfaces in an active or open form, thus implying a displacement of the helical lid away from the active site. A study of the influence of substrate and diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. The interfacial activation is not supported by zwitterionic vesicles or by large anionic vesicles of 100 nm diameter, although TlL binds to these interfaces. Using a combination of fluorescence-based techniques applied to several mutants of TlL with different tryptophan residues we have shown that TlL binds to phospholipid vesicles in different forms rendering different catalytic activities, and that the open lid conformation is achieved and stabilized by a combination of electrostatic and hydrophobic interactions between the enzyme's lipid-binding face and the interface.     

Detergent-induced conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy

Detergent (pentaoxyethylene octyl ether, C(8)E(5))-induced conformational changes of Humicola lanuginosa lipase (HLL) were investigated by stationary and time-resolved fluorescence intensity and anisotropy measurements. Activation of HLL is characterized by opening of a surface loop (the "lid") residing directly over the enzyme active site. The interaction of HLL with C(8)E(5) increases fluorescence intensities, prolongs fluorescence lifetimes, and decreases the values of steady-state anisotropy, residual anisotropy, and the short rotational correlation time. Based on these data, we propose the following model. Already below critical micellar concentration (CMC) the detergent can intercalate into the active site accommodating cleft, while the lid remains closed. Occupation of the cleft by C(8)E(5) also blocks the entry of the monomeric substrate, and inhibition of catalytic activity at [C(8)E(5)] less than or equal to CMC is evident. At a threshold concentration close to CMC the cooperativity of the hydrophobicity-driven binding of C(8)E(5) to the lipase increases because of an increase in the number of C(8)E(5) molecules present in the premicellar nucleates on the hydrophobic surface of HLL. These aggregates contacting the lipase should have long enough residence times to allow the lid to open completely and expose the hydrophobic cleft. Concomitantly, the cleft becomes filled with C(8)E(5) and the "open" conformation of HLL becomes stable.     

Phospholipases and their industrial applications

Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A₁, A₂, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions—white biotechnology is a strong alternative. An erratum to this article can be found at     

A comparative study of activity and apparent inhibition of fungal β‐glucosidases

β‐Glucosidases (BGs) from Aspergillus fumigates, Aspergillus niger, Aspergillus oryzae, Chaetomium globosum, Emericella nidulans, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. Global non‐linear regression of several reactions, with or without added glucose, to a product inhibition equation enabled the concurrent derivation of the kinetic parameters k_cat, K_m, and the apparent product inhibition constant _appK_i for each of the enzymes. A more simple fit is not advisable to use as the determined _appK_i are in the same range as their K_m for some of the tested BGs and produced glucose would in these cases interfere. The highest value for k_cat was determined for A. fumigatus (768 s^?1) and the lowest was a factor 9 less. K_m varied by a factor of 3 with the lowest value determined for C. globosum (0.95 mM). The measured _appK_i varied a factor of 15; the hydrolytic activity of N. crassa being the most resistant to glucose with an apparent product inhibition constant of 10.1 mM. Determination of _appK_i using cellobiose as substrate is important as it reflects to what extent the different BGs are hydrolytically active under industrial conditions where natural substrates are hydrolyzed and the final glucose concentrations are high. Biotechnol. Bioeng. 2010;107: 943–952. © 2010 Wiley Periodicals, Inc.     

Conditionally immortalised neural stem cells promote functional recovery and brain plasticity after transient focal cerebral ischaemia in mice

Cell therapy has enormous potential to restore neurological function after stroke. The present study investigated effects of conditionally immortalised neural stem cells (ciNSCs), the Maudsley hippocampal murine neural stem cell line clone 36 (MHP36), on sensorimotor and histological outcome in mice subjected to transient middle cerebral artery occlusion (MCAO).Adult male C57BL/6 mice underwent MCAO by intraluminal thread or sham surgery and MHP36 cells or vehicle were implanted into ipsilateral cortex and caudate 2 days later. Functional recovery was assessed for 28 days using cylinder and ladder rung tests and tissue analysed for plasticity, differentiation and infarct size.MHP36-implanted animals showed accelerated and augmented functional recovery and an increase in neurons (MAP-2), synaptic plasticity (synaptophysin) and axonal projections (GAP-43) but no difference in astrocytes (GFAP), oligodendrocytes (CNPase), microglia (IBA-1) or lesion volumes when compared to vehicle group.This is the first study showing a potential functional benefit of the ciNSCs, MHP36, after focal MCAO in mice, which is probably mediated by promoting neuronal differentiation, synaptic plasticity and axonal projections and opens up opportunities for future exploitation of genetically altered mice for dissection of mechanisms of stem cell based therapy.     

Flow cytometric analysis of total protein content and size distributions of recombinantSaccharomyces cerevisiae

The total protein content and cell size distribution of recombinantSaccharomyces cerevisiae cells were analyzed by flow cytometry. The recombinant strain containing a regulatableSUC2 promoter and the host strain were compared when grown under similar conditions in a batch culture. Recombinant and host cells maintained similar size and total protein content while cloned-gene expression was repressed by glucose levels greater than 0.2% (w/v). Following derepression, recombinant cells demonstrated a mean total protein content and mean cell size 1.5–2 times greater than that of the host cells. In addition, these simple flow cytometric measurements of the changes in cell size and total protein content were found to closely follow diauxic growth ofSaccharomyces cerevisiae in batch culture.     

Neuronal Nicotinic Acetylcholine Receptor Modulators Reduce Sugar Intake

Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption.