Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group^1-3. JoVE video articles describing the method are available^1,4. The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group^5,6. The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.     

Glutamate receptors of the A5 region modulate cardiovascular responses evoked from the dorsomedial hypothalamic nucleus and perifornical area

To assess the possible function of glutamate in the interaction between the dorsomedial hypothalamic nucleus-perifornical area (DMH-PeF) and the A5 pontine region (A5), cardiovascular and respiratory changes were studied in response to electrical stimulation of the DMH-PeF (1 ms pulses, 30–50 μA given at 100 Hz for 5 s) before and after the microinjection of kynurenic acid (non-specific glutamate receptor antagonist; 50 nl, 5 nmol), MK-801 (NMDA receptor antagonist; 50 nl, 50 nmol), CNQX (non-NMDA receptor antagonist; 50 nl, 50 nmol) or MCPG (metabotropic glutamate receptor antagonist; 50 nl, 5 nmol) within the A5 region. DMH-PeF electrical stimulation elicited a pressor (p <?0.001) and tachycardic response (p <?0.001) which was accompanied by an inspiratory facilitation characterised by an increase in respiratory rate (p <?0.001) due to a decrease in expiratory time (p <?0.01). Kynurenic acid within the A5 region decreased the tachycardia (p <?0.001) and the intensity of the blood pressure response (p <?0.001) to DMH-PeF stimulation. After the microinjection of MK-801 and CNQX into the A5 region, the magnitude of the tachycardia and the pressor response were decreased (p <?0.05 and p <?0.01; p <?0.001 and p <?0.05, respectively). After MCPG microinjection into the A5 region, a decrease in the tachycardia (p <?0.001) with no changes in the pressor response was observed during DMH-PeF stimulation. The respiratory response elicited by DMH-PeF stimulation was not changed after the microinjection of kynurenic acid, MK-801, CNQX or MCPG within the A5 region. These results suggest that A5 region glutamate receptors play a role in the cardiovascular response elicited from the DMH-PeF. The possible mechanisms involved in these interactions are discussed.     

Controlled in meso phase crystallization--a method for the structural investigation of membrane proteins

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time.     

Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol

D-Ribitol, a five–carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27–30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid–Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol–KLH–Sepharose CL-6B resulted in pure ribitol–specific antibodies (~45–50 μg/mL). The affinity constant of ribitol antibodies was found to be 2.9?×?10⁷ M^?1 by non-competitive ELISA. Ribitol antibodies showed 100 % specificity towards ribitol, ~800 % cross–reactivity towards riboflavin, 10–15 % cross–reactivity with sorbitol, xylitol and mannitol, and 5–7 % cross–reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4 %) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.     

Enhanced enzymatic cellulose degradation by cellobiohydrolases via product removal

Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5–6.5, and CBHII: 46 °C, pH 4.8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion in the parallel batch reaction was only ~5 % by weight. Also, a synergistic effect, achieving ~27 % substrate conversion, was obtained by addition of endo-1,4-β-d-glucanase. The synergistic effect was only obtained with product removal. By using pure, monoactive enzymes, the work illustrates the profound gains achievable by intermittent product removal during cellulose hydrolysis.     

Paenibacillus thermophilus sp. nov., a novel bacterium isolated from a sediment of hot spring in Fujian province, China

A Gram-positive, thermophilic, strictly aerobic bacterium, designated WP-1^T, was isolated from a sediment sample from a hot spring in Fujian province of China and subjected to a polyphasic taxonomic study. Cells of strain WP-1^T were rods (~0.6–0.8 × 2.5–3.5 μm) and motile by means of peritrichous flagella. Endospores were ellipsoidal in terminal or subterminal positions. Strain WP-1^T grew at 37–60 °C (optimum 42–45 °C), 0–3 % NaCl (optimum 1 %, w/v) and pH 3.0–9.0 (optimum pH 6.5–7.0). The predominant menaquinone was MK-7. The major fatty acids were anteiso-C_15:0, iso-C_16:0, C_16:0 and anteiso-C_17:0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, two glycolipids, two unidentified phospholipids and two unknown polar lipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid (meso-DAP). The G + C content of the genomic DNA was 52.5 %. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain WP-1^T is a member of the genus Paenibacillus and exhibited sequence similarity of 99.3 % to Paenibacillus macerans DSM 24^T and both strains represented a separate lineage from all other Paenibacillus species. However, the level of DNA–DNA relatedness between strain WP-1^T and P. macerans DSM 24^T was 34.0 ± 4.7 %. On the basis of phylogenetic, physiological and chemotaxonomic analysis data, strain WP-1^T is considered to represent as a novel species of the genus Paenibacillus, for which the name Paenibacillus thermophilus sp. nov., is proposed, with the type strain WP-1^T (=DSM 24746^T = JCM 17693^T = CCTCC AB 2011115^T).     

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.     

Enhanced activity of meso-secondary alcohol dehydrogenase from Klebsiella species by codon optimization

Meso-secondary alcohol dehydrogenases (meso-SADH) from Klebsiella oxytoca KCTC1686 and Klebsiella pneumoniae KCTC2242 were codon optimized and expressed in Escherichia coli W3110. The published gene data of K. pneumoniae NTUH-K2044 (NCBI accession number AP006725), K. pneumoniae 342 (NCBI accession number CP000964), and K. pneumoniae MGH 78578 (NCBI accession number CP000647), were compared with the meso-SADH sequences of each strain, respectively. Codon-optimized meso-SADH enzymes of K. oxytoca and K. pneumoniae showed approximately twofold to fivefold increased enzyme activities for acetoin reduction over native enzymes. The highest activities for each strain were obtained at 30–37 °C and pH 6–7 (yielding 203.1 U/mg of protein and 156.5 U/mg of protein, respectively). The increased enzyme activity of the codon-optimized enzymes indicated that these modified enzymes could convert acetoin into 2,3-butanediol with a high yield.     

Physico-kinetic and functional features of a novel β-glucosidase isolated from milk thistle (Silybum marianum Gaertn.) flower petals

A highly abundant β-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and β-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (~16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with K_m and V_max values of 0.25 mM and 5.35 μkat.mg^?1 enzyme protein. Thus, the enzyme had a very high (292,000 M^?1.s^?1) catalytic efficiency (K_cat/K_m). Thermal catalytic optimum of enzyme was 40 °C with activation energy value 8.26 kCal.Mol^?1. The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent K_i 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications.     

Characterization of an NAD(P)+-dependent meso-diaminopimelate dehydrogenase from Thermosyntropha lipolytica

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP⁺-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP⁺ and NAD⁺ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5–9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.     

Expression of a lipase on the cell-surface of Escherichia coli using the OmpW anchoring motif and its application to enantioselective reactions

Microbial-surface display is the expression of proteins or peptides on the surface of cells by fusing an appropriate protein as an anchoring motif. Here, the outer membrane protein W (OmpW) was selected as a fusion partner for functional expression of Pseudomonas fluorescence SIK W1 lipase (TliA) on the cell-surface of Escherichia coli. Localization of the truncated OmpW-TliA fusion protein on the cell-surface was confirmed by immunoblotting and functional assay of lipase activity. Enantioselective hydrolysis of rac-phenylethyl butanoate by the displayed lipase resulted in optically active (R)-phenyl ethanol with 96 % enantiomeric excess and 44 % of conversion in 5 days. Thus, a small outer membrane protein OmpW, is a useful anchoring motif for displaying an active enzyme of ~50 kDa on the cell-surface and the surface-displayed lipase can be employed as an enantioselective biocatalyst in organic synthesis.     

Comparative studies of water permeability of red blood cells from humans and over 30 animal species: an overview of 20 years of collaboration with Philip Kuchel

NMR measurements of the diffusional permeability of the human adult red blood cell (RBC) membrane to water (P _d) and of the activation energy (E _a,d) of the process furnished values of P _d ~ 4 × 10^?3 cm/s at 25 °C and ~6.1 × 10^?3 cm/s at 37 °C, and E _a,d ~ 26 kJ/mol. Comparative NMR measurements for other species showed: (1) monotremes (echidna and platypus), chicken, little penguin, and saltwater crocodile have the lowest P _d values; (2) sheep, cow, and elephant have P _d values lower than human P _d values; (3) cat, horse, alpaca, and camel have P _d values close to those of humans; (4) guinea pig, dog, dingo, agile wallaby, red-necked wallaby, Eastern grey kangaroo, and red kangaroo have P _d values higher than those of humans; (5) mouse, rat, rabbit, and “small and medium size” marsupials have the highest values of P _d (>8.0 × 10^?3 cm/s at 25 °C and >10.0 × 10^?3 cm/s at 37 °C). There are peculiarities of E _a,d values for the RBCs from different species. The maximum inhibition of diffusional permeability of RBCs induced by incubation with p-chloromercuribenzene sulfonate varied between 0 % (for the chicken and little penguin) to ~50 % (for human, mouse, cat, sheep, horse, camel, and Indian elephant), and ~60–75 % (for rat, guinea pig, rabbit, dog, alpaca, and all marsupials). These results indicate that no water channel proteins (WCPs) or aquaporins are present in the membrane of RBCs from monotremes (echidna, platypus), chicken, little penguin and saltwater crocodile whereas WCPs from the membranes of RBCs from marsupials have peculiarities.     

Growth and physiochemical responses of <Emphasis Type="Italic">Camelina sativa</Emphasis> L. under UV-C stress

A study was conducted to estimate the effect of UV-C (200–280 nm) radiation stress on growth and physiochemical responses of Camelina sativa L. cv. Calina (EC643910; a potential bio-fuel crop) for its possible mass multiplication at high-altitude under high radiations. The germination percentage in terms of radicle protrusion and opening of cotyledonary leaves significantly decreased 13.98 and 27.8 %, respectively, as compared to control. However, no significant change was observed in growth parameters including root and shoot lengths and fresh biomass. The relative membrane leakage rate and lipid peroxidation as a malondialdehyde contents significantly increased with the value of ~99 % and 0.17 mM g^?1 FW, respectively, under UV-C stress. Also, the proline, glycine betaine and total soluble sugars contents increased by ~330, ~440, ~144 %, respectively, as compared to control. Among non-enzymatic antioxidants, the ascorbic acid and total phenol contents significantly increased by ~284 and ~537 %, respectively, as compared to control. Likewise, the activity of antioxidant enzyme, ascorbate peroxidase, guaiacol peroxidase and catalase increased under UV-C stress with the value of 1.03, 0.05 and 0.06 µmol mg protein^?1 min^?1, respectively. In addition, the chlorophyll a, b and total (a + b) contents decreased by ~180, ~151 and ~147 %, respectively, as compared to control. In contrast, the total carotenoids and anthocyanin contents increased by ~160 and ~184 %, respectively. Our findings suggest the adaptive growth and physiochemical responses of C. sativa under UV-C stress. Therefore, it may be recommended for large-scale cultivation at high-altitude under intense natural radiations for future bio-fuel production.     

Assembly of photosynthetic apparatus in Rhodobacter sphaeroides as revealed by functional assessments at different growth phases and in synchronized and greening cells

The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0–4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10⁴ s^?1) and yield (F _v/F _max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10⁴ s^?1). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.     

Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35

Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured β-barrel membrane proteins, like outer membrane proteins OmpA from Escherichia coli and FomA from Fusobacterium nucleatum, can be folded by dilution of the denaturant urea in the presence of amphipol A8-35. Here, the folding kinetics and stability of OmpA in A8-35 have been investigated. Folding is well described by two parallel first-order processes, whose half-times, ~5 and ~70 min, respectively, are independent of A8-35 concentration. The faster process contributed ~55–64 % to OmpA folding. Folding into A8-35 was faster than into dioleoylphosphatidylcholine bilayers and complete at ratios as low as ~0.17 g/g A8-35/OmpA, corresponding to ~1–2 A8-35 molecules per OmpA. Activation energies were determined from the temperature dependence of folding kinetics, monitored both by electrophoresis, which reports on the formation of stable OmpA tertiary structure, and by fluorescence spectroscopy, which reflects changes in the environment of tryptophan side chains. The two methods yielded consistent estimates, namely ~5–9 kJ/mol for the fast process and ~29–37 kJ/mol for the slow one, which is lower than is observed for OmpA folding into dioleoylphosphatidylcholine bilayers. Folding and unfolding titrations with urea demonstrated that OmpA folding into A8-35 is reversible and that amphipol-refolded OmpA is thermodynamically stable at room temperature. Comparison of activation energies for folding and unfolding in A8-35 versus detergent indicates that stabilization of A8-35-trapped OmpA against denaturation by urea is a kinetic, not a thermodynamic phenomenon.     

Cloning,expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K _m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation–reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe²⁺ and for (3S/3R)-acetoin reduction in the presence of Mn²⁺, while several cations inhibited its activity, particularly Fe²⁺ and Fe³⁺ for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .     

NaCl increases the activity of the plasma membrane H+-ATPase in C3 halophyte Suaeda salsa callus

Suaeda salsa calli treated with different concentrations of NaCl were used to examine the response of the plasma membrane (PM) H⁺-ATPase to NaCl and its role in salt tolerance. The optimum concentration of NaCl for growth of the calli was 50 mM, while growth was significantly inhibited at 250 mM NaCl. The ion and organic solute contents of calli increased with increasing NaCl. Activity of the PM H⁺-ATPase increased when the calli were treated with NaCl over a certain concentration range (0–150 mM NaCl). However, the activity reached its maximum with 150 mM NaCl. Immunoblotting analysis of the PM H⁺-ATPase protein from calli cultures with anti-Zea mays H⁺-ATPase serum (monoclonal 46E5B11D5) identified a single polypeptide of ~90 kDa. The peptide levels increased in the calli treated with NaCl at 150 mM NaCl compared to control, but the increase at 50 mM NaCl was less pronounced. Northern blot analysis showed that the expression of the PM H⁺-ATPase also increased after the calli were treated with NaCl. These results suggest that the increase in PM H⁺-ATPase activity is due to both an increase in the amount of PM H⁺-ATPase protein and an up-regulation of the PM H⁺-ATPase gene, which is involved in the salt tolerance of S. salsa calli.     

Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Arylsulfatase

To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t _0.5) at 37 °C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t _0.5 of ~6.0 days in 50 mM HEPES, but ~42 days in 10 mM sodium borate with >85 % activity after 15 days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t _0.5 of PAAS was ~14 days in 50 mM Tris–HCl, and ~21 days in 10 mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14 days at 37 °C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.     

Enhancement of Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.     

Quantification of soybean seed germination response to seed deterioration under PEG-induced water stress using hydrotime concept

This experiment was arranged to investigate the ability of hydrotime model (θ_H) for estimating soybean seed germination (cv. ‘JK’) under different accelerated aging periods (AAP, 0, 24, 48, and 72 h) at each of the following water potentials (ψ, 0, ??0.12, ??0.24, and ??0.36 MPa). Results indicated that both germination percentage (GP) and germination rate (GR) significantly influenced by ψ, AAP, and their interactions (P?<?0.01). GP and GR decreased by 62.6 and 47.3% with longer AAP from 0 to 72 h and by 90.7 and 81.5% with lower ψ from zero to ??0.36 MPa as compared to the control, respectively. Therefore, the effect of ψ on GP and GR was more than AAP. The θ_H value was constant (~?6.71 MPa h^?1) till 50.6 h AAP and then linearly declined with the rate of 0.1545 MPa h^?1 per hour increase in AAP until 72 h (~?50% lower than its initial value). The ψ_b(50) value was ? 0.343 MPa in the control and then increased (became more positive) by ~?70% until 72 h AAP (? 0.104 MPa). In general, GP and GR of soybean declined with the increasing ψ_b(50) which can be due to cell membrane damage and reduce the activity of enzymes and organelles during AAP. Based on our findings, the θ_H model could describe well these relationships and their parameters can nicely be used for simulating soybean seed germination under this condition.