Immobilization of β-glucosidase on Eupergit C for Lignocellulose Hydrolysis

β-Glucosidase is frequently used to supplement cellulase preparations for hydrolysis of cellulosic and lignocellulosic substrates in order to accelerate the conversion of cellobiose to glucose. Typically, commercial cellulase preparations are deficient in this enzyme and accumulation of cellobiose leads to product inhibition. This study evaluates the potential for recycling β-glucosidase by immobilization on a methacrylamide polymer carrier, Eupergit C. The immobilized β-glucosidase had improved stability at 65 °C, relative to the free enzyme, while the profile of activity versus pH was unchanged. Immobilization resulted in an increase in the apparent K_m from 1.1 to 11 mm and an increase in V_max from 296 to 2430 μmol mg⁻¹ min⁻¹. The effect of immobilized β-glucosidase on the hydrolysis of cellulosic and lignocellulosic substrates was comparable to that of the free enzyme when used at the same level of protein. Operational stability of the immobilized β-glucosidase was demonstrated during six rounds of lignocellulose hydrolysis. Received 22 August 2005; Revisions requested 20 September 2005; Revisions received 8 November 2005; Accepted 10 November 2005     

Formation of Multilayers at the Interface of Oil-in-Water Emulsion via Interactions between Lactoferrin and β-Lactoglobulin

The interactions between negatively charged β-lactoglobulin and the positively charged lactoferrin at the droplet surface to form a multi-protein surface layer were examined. Addition of lactoferrin to the aqueous phase of emulsions formed with β-lactoglobulin at pH 7.0 caused an increase in the ζ-potential of emulsion droplets, and the ζ-potential became positive as the concentration of added lactoferrin was higher than 1% in the system. It is found that lactoferrin binds to adsorbed β-lactoglobulin at droplet surface probably via electrostatic interactions. The amount of lactoferrin at interface increased with increasing the concentration of added lactoferrin, but it decreased with a decrease in the pH. No lactoferrin was observed at interface at pH 3 and 4. By contrast, when β-lactoglobulin was added in the emulsions formed with lactoferrin at pH 7.0, the ζ-potential of emulsions changed from positive to negative as the concentration of added β-lactoglobulin increased. The amount of β-lactoglobulin at surface increased correspondingly with increasing the concentration of added β-lactoglobulin. However, in this case, β-lactoglobulin remained bound at interface even at pH 3 and 4 where both lactoferrin and β-lactoglobulin are positively charged. The association of lactoferrin or β-lactoglobulin with the surface proteins that have oppositely charge is probably mainly through electrostatic interactions between the two proteins. It appears that alternative layers of these proteins could be created at the droplet surface.     

Properties of Laccases Produced by Pycnoporus sanguineus Induced by 2,5-xylidine

Two isoforms of laccase produced from the culture supernatant of Pycnoporus sanguineus were partially purified by phenyl-Sepharose chromatography. Molecular masses of the enzymes were 80 kDa (Lac I) and 68 kDa (Lac II). Optimum activity of Lac I was at pH 4.8 and 30 °C, and Lac II was at pH 4.2 and 50 °C over 5 min reaction. The K_m values of enzymes toward syringaldazine were 10 μm (Lac I) and 8 μm (Lac II). Sodium azide inhibited Lac I (85%) and Lac II (75%) activities. Revisions requested 30 November 2005; Revisions received 26 January 2006     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Formation of Non-Native ??-Lactoglobulin during Heat-Induced Denaturation

A mechanism describing the denaturation and aggregation behavior during heat-treatment of pure β-lactoglobulin and β-lactoglobulin in whey protein isolate (WPI) under selected conditions (20 to 90 gL⁻¹ in water at pH 7.0, 78 °C) is presented. A combination of reversed-phase and gel permeation chromatography was used to study the disappearance of native β-lactoglobulin and the formation of non-native intermediates in the aggregation process. The mean reaction order for pure β-lactoglobulin and β-lactoglobulin in WPI were the same, 1.4. While the rate of β-lactoglobulin denaturation was greater in WPI there was less aggregation compared to that of pure β-lactoglobulin. More of the β-lactoglobulin in WPI remained in a non-native monomer intermediate state after 30 min of heating. After an initial lag period, during which non-native monomers appeared, aggregates formed and rapidly reached a plateau in terms of their size. These aggregates were visualized using atomic force microscopy. There was no significant effect of protein concentration on either aggregate size or the number of exposed sulfhydryls in the heated solutions.     

Aluminium Chloride Enhances Colchicine Production in Root Cultures of Gloriosa superba

Root cultures of Gloriosa superba were treated with 5 mm methyl jasmonate and 125 μm AlCl₃ which enhanced the intracellular colchicine content of the roots by 50-fold and 63-fold, respectively. Ten millimolar of CaCl₂ and 1 mm CdCl₂ enhanced biomass significantly (7- to 8.6-fold, respectively) while maximum release of colchicine into the medium was obtained with 10 mm CdCl₂. Casein hydrolysate, yeast extract and silver nitrate had no significant effect on growth and colchicine accumulation in root cultures. Revisions requested 2 November 2005; Revisions received 9 January 2006     

Betaine Tripled the Volumetric Productivity of d(-)-lactate by Escherichia coli Strain SZ132 in Mineral Salts Medium

Osmotic stress restricts glycolytic flux, growth (rate and yield), d-lactate productivity, and d-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mm betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of d-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l⁻¹ h⁻¹; 0.8 to 2.3 g l⁻¹ h⁻¹). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during d-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol d-lactate (90 g) per 100 g sugar (glucose or sucrose). Revisions requested 17 January 2006; Revisions received 7 February 2006     

Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice

We are studying the tissue-specific expression of the sheep milk-whey protein gene, β-lactoglobulin. We have used sequences derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology in transgenic sheep as a means of protein production. In the present study, a series of β-lactoglobulin hybrid genes and β-lactoglobulin minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5′ flanking sequences of β-lactoglobulin fused to a genomic minigene encoding human α-antitrypsin (α₁AT) was expressed much more efficiently than an α₁AT-cDNA construct containing the same β-lactoglobulin segment. Similarly, the intact β-lactoglobulin gene was expressed more efficiently than the corresponding intronless β-lactoglobulin minigene. This effect was not seen in transient expression expriments in baby hamster kidney cells when β-lactoglobulin-α₁AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing, since the inclusion of the first β-lactoglobulin intron into cDNA constructs encoding human α₁AT or β-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements within introns may interact with the upstream 5′ flanking sequences of β-lactoglobulin and enable the latter to function efficiently in the mammary gland of transgenic mice.     

Purification and biochemical characterization of a transglucosilating β-glucosidase of <Emphasis Type="Italic">Stachybotrys</Emphasis> strain

The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, K _m and V _max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min⁻¹ ml⁻¹. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.     

Protein Kinase C and Regulatory Volume Decrease in Mudpuppy Red Blood Cells

This study examined whether protein kinase C (PKC) stimulates K⁺ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K⁺ efflux (gramicidin is a cationophore that was used to ensure a high K⁺ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K⁺ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K⁺ loss during RVD is regulated, at least in part, by protein kinase C. Received: 20 January 1998/Revised: 2 September 1998     

Isolation of a Fusion Protein Containing the Antigenic Domain 1 of Human Cytomegalovirus Glycoprotein B and its Application in ELISA Tests

The glycoprotein B (gB) of human cytomegalovirus represents a dominant antigen for the humoral immune response. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids for antibody binding. In this study, this domain was expressed in Escherichia coli as a fusion protein with β-galactosidase but yielded insoluble protein aggregates as inclusion bodies. To recover the fusion protein, inclusion bodies were solubilized by two extractions with urea 8 m and the fusion protein then isolated using gel filtration chromatography. After confirmation of fusion protein antigenicity by Western blotting, the purified product was used as the capturing antigen in an enzyme-linked immunosorbent assay (ELISA) to determine the presence of viral antibodies in serum samples of pregnant women. A cut-off point of approximately 0.2 absorbance units could discriminate the results of seropositive from seronegative pregnant women. The data indicates the potential usefulness of the fusion protein for the development of immunoassay for detection of the HCMV antibodies. Received 22 September 2005; Revisions requested 10 October 2005; Revisions received 26 October 2005; Accepted 31 October 2005     

Effect of Gibberellin on Growth, Protein Secretion, and Starch Accumulation in Maize Endosperm Suspension Cells

The physiologic effect of gibberellins (GA) in seed development is poorly understood. We examined the effect of gibberellic acid (GA₃) on growth, protein secretion, and starch accumulation in cultured maize (Zea mays L.) endosperm suspension cells. GA₃ (5 and 30 μm) increased the fresh weight, dry weight, and protein content of the cultured cells, but the effect of GA₃ at 50 μm was not significantly different. However, the protein content in the culture medium was increased by these three concentrations of GA₃. The effect of GA₃ on the amount of cellular structural polysaccharides was not significant, but GA₃ had a dramatic effect on the starch content. At 5 μm, GA₃ caused an increase in the starch content, but at 50 μm the starch accumulation was reduced. Chlorocholine chloride (CCC), an inhibitor of GA biosynthesis, significantly increased the starch content and decreased the structural polysaccharide content of the cultured cells. The effects of CCC at 500 μm on the starch and polysaccharide content were partially reversed by 5 μm GA₃ applied exogenously. Based on these results we suggest that GA does not favor starch accumulation in the cell cultures and that the addition of lower concentrations of GA₃ in the medium may provide an improved balance among the endogenous GA in the cultured cells. Received October 31, 1995; accepted March 25, 1997     

Influence of auxins on growth ofClaviceps purpurea (Fries) tulasne in saprophytic cultures

A 10⁻³ m β-indolebutyric and 2,4-dichlorophenoxyacetic acid concentration stimulated the fresh weight increase and dry matter production in the mycelium of staticClaviceps purpurea cultures to 170–180% of the control value. Higher concentrations severely inhibited growth. α-Naphthylacetic acid was less active. β-Indoleacetic acid differed markedly from the other auxins, as it stimulated the fresh weight increase, but at the same time inhibited dry matter production. In submerse cultivation, the differences between the test auxins disappeared; none of them stimulated growth and only inhibition of growth, by concentrations of over 10⁻⁵–10⁻⁴ m, was observed. The used strain did not produce alkaloids in saprophytic cultures, even in the presence of auxins.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Enhancement of 1,3-propanediol Production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> with Fumarate Addition

Addition of 5 mm fumarate to cultures of Klebsiella pneumoniae enhanced the rate of glycerol consumption and the production of 1,3-propanediol (PDO). Compared to the control, the activity of glycerol dehydrogenase increased by 35, 33 and 46%, the activity of glycerol dehydratase increased by 160, 210 and 115%, and the activity of 1,3-propanediol oxidoreductase increased by 25, 39 and 85% when, respectively, 5, 15 and 25 mm fumarate were provided. At the same time, the ratio of NAD⁺ to NADH decreased by 20, 23 and 29%. Using a 5 l bioreactor with 5 mM fumarate addition, the specific rate of glycerol consumption and the productivity of PDO was 30 mmol/l h and 17 mmol/l h, respectively, both increased by 35% over the control. Revisions requested 15 July 2005; Revisions received 30 August 2005     

Ion Channels Formed by NB,an Influenza B Virus Protein

The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions with unequal concentrations (150–500 mm cis, 50 mm trans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2–3 mm), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately 10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable to sodium than to chloride ions (P_Na/P_Cl∼ 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium potential indicating that the channel had become more permeable to chloride than to sodium ions (P_Cl/P_Na∼ 4). It was concluded that, at normal pHs, NB forms cation-selective channels. Received: 6 March 1995/Revised: 17 November 1995     

Production of Bioactive Human β-defensin-3 in Escherichia coli by Soluble Fusion Expression

A codon optimized mature human β-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l⁻¹, i.e. 0.21 g mature HBD3 l⁻¹. Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli. Revisions requested 13 December 2005; Revisions received 24 January 2006