Identification of aryl-phospho-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidases in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

Four aryl-phospho--d-glucosidases were identified in Bacillus subtilis by using 4-methylumbelliferyl-phospho--d-glucopyranoside as a substrate. Two of these enzymes are the products of the bglA and bglH genes, previously suggested to encode aryl-phospho--d-glucosidases, while the other enzymes are encoded by the yckE and ydhP genes. Together, these four genes account for >99.9% of the glucosidase activity in B. subtilis on aryl-phospho--d-glucosides. yckE was expressed at a low and constant level during growth, sporulation, and spore germination, and was not induced by aryl--d-glucosides. ydhP was also not induced by aryl--d-glucosides. However, while ydhP was expressed at only a very low level in exponential-phase cells and germinating spores, this gene was expressed at a higher levels upon entry into the stationary phase of growth. Strains lacking yckE or ydhP exhibited no defects in growth, sporulation, or spore germination or in growth on aryl--d-glucosides. However, a strain lacking bglA, bglH and yckE grew poorly if at all on aryl--d-glucosides as the sole carbon source.Abbreviations MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--d-glucopyranoside - MUGal 4-Methylumbelliferyl--d-galactopyranoside - MUG-P 4-Methylumbelliferyl--d-glucopyranoside-6-phosphate     

Cyclitols affect accumulation of α-<Emphasis Type="SmallCaps">d</Emphasis>-galactosides in developing <Emphasis Type="Italic">Vicia</Emphasis> seeds

The mechanism preferentially regulating accumulation of raffinose family oligosaccharides (RFOs) or galactosyl cyclitols in legume seeds still remains unknown. The broad range of raffinose family oligosaccharides and galactosyl pinitols in the composition of seeds of Vicia genus gives researchers an exceptional opportunity for investigations on relationships in biosynthesis of both types of α-d-galactosides. Feeding explants of Vicia species radically different in the composition of RFOs and galactosyl pinitols with basic galactose acceptors, sucrose (for RFOs) or cyclitols (for galactosyl cyclitols) can be a helpful method for assessment of their regulatory role in accumulation of α-d-galactosides in seeds. Garden vetch (Vicia sativa L.) seeds, naturally accumulating RFOs, demonstrated an ability to take up and use exogenously applied d-pinitol and d-chiro-inositol for synthesis of their mono-, di- and tri-galactosides. Together with the accumulation of new galactosides, the concentration of RFOs decreased. In fine-leaved (Vicia tenuifolia Roth.) vetch seeds such a remarkably high concentration of galactosyl pinitols (GPs) was discovered that they nearly replaced RFOs, which is unique among legumes. If the accumulation of both types of galactosides is correlated with concentration of galactose acceptors, elevated levels of sucrose or myo-inositol should promote accumulation of RFOs, instead of GPs. Unexpectedly, feeding fine-leaved vetch raceme explants with myo-inositol or sucrose promoted accumulation of GPs, but not of RFOs. Our comparison of accumulation and biosynthesis of both types of galactosides (RFOs and GPs) throughout development and maturation of seeds from fine-leaved vetch has indicated that preferential accumulation of GPs is associated with the drying of seeds during maturation. Different patterns in activities of enzymes engaged in RFOs’ biosynthetic pathway and galactosyltransferases involved in biosynthesis of GPs indicated that distinct forms of enzymes can operate in both pathways. The feeding of explants with d-chiro-inositol causes accumulation of fagopyritols B1 in seeds of both Vicia species, which suggests presence of the same or a similar form of galactinol synthase. Accumulation of fagopyritols in fine-leaved vetch seeds did not affect accumulation of RFOs or galactosyl pinitols.     

Growth inhibitory activity of ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranosyl ester and ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidase

Exogenously applied ABA-β-d-glucopyranosyl ester (ABA-GE) inhibited shoot growth of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), Digitaria sanguinalis L., timothy (Pheleum pratense L.) and ryegrass (Lolium multiflorum Lam.) seedlings at concentrations greater than 0.1 μM. The growth inhibitory activity of ABA-GE on these shoots was 26–40% of that of (+)-ABA. ABA-β-d-glucosidase activities in these seedlings were 11–31 nmol mg⁻¹ protein min⁻¹. These results suggests that exogenously applied ABA-GE may be absorbed by plant roots and hydrolyzed by ABA-β-d-glucosidase, and liberated free ABA may induce the growth inhibition in these plants. Thus, although ABA-GE had been thought to be physiologically inactive ABA conjugate, ABA-GE may have important physiological functions rather than an inactive conjugated ABA form.     

The α-<Emphasis Type="SmallCaps">l</Emphasis>-fucosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.     

Purification and Characterization of a Novel β-<Emphasis Type="SmallCaps">d</Emphasis>-Galactosides-Specific Lectin from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography. HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal.     

Endogeneous β-<Emphasis Type="SmallCaps">d</Emphasis>-xylosidase and α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase activity in flax seed mucilage

Flax seed mucilage (FM) contains a mixture of highly doubly substituted arabinoxylan as well as rhamnogalacturonan I with unusual side group substitutions. Treatment of FM with a GH11 Bacillus subtilis XynA endo 1,4-β-xylanase (BsX) gave limited formation of reducing ends but when BsX and FM were incubated together on different wheat arabinoxylan substrates and birchwood xylan, significant amounts of xylose were released. Moreover, arabinose was released from both water-extractable and water-unextractable wheat arabinoxylan. Since no xylose or arabinose was released by BsX addition alone on these substrates, nor without FM or BsX addition, the results indicate the presence of endogenous β-d-xylosidase and α-l-arabinofuranosidase activities in FM. FM also exhibited activity on both p-nitrophenyl α-l-arabinofuranoside (pNPA) and p-nitrophenyl β-d-xylopyranoside (pNPX). Based on K _M values, the FM enzyme activities had a higher affinity for pNPX (K _M 2 mM) than for pNPA (K _M 20 mM).     

In-fusion expression and characterization of <Emphasis Type="Italic">β</Emphasis>-xylanase and <Emphasis Type="Italic">β</Emphasis>-1,3-1,4-glucanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower K_n higher k_cat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.     

Immobilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase activity for <Emphasis Type="SmallCaps">d</Emphasis>(−)-tartaric acid production

Immobilization of cis-epoxysuccinate hydrolase-containing E. coli for d(−)-tartaric acid production was screened by various methods. The highest recovery of activity was obtained by entrapment in κ-carrageenan gel. 23.6 g biomass/l and 43.4 g κ-carrageenan/l were the best immobilization conditions optimized by response surface methodology with 83% yield (114 U/g). Cell autolysis was observed after immobilization. Immobilized cells showed high pH (5–10) stability, thermal (up to 65°C) stability, conversion rate (>99.5%), enantioselectivity (ee > 99.6%), and were less affected by metal ions and surfactants compared with free cells. Conversion rate for immobilized cells preserved 93% after 10 repeated batches (5% for free cells).     

High-level expression of a truncated 1,3-1,4-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by optimization of codons and fermentation

1,3-1,4-β-d-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the α-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48–49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1–3 and 100% methanol was used on days 4–6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-β-d-glucanase constituted ~90% of the total secreted protein, reaching up to 3 g l⁻¹ in the medium.     

Efficient synthesis of 6-<Emphasis Type="Italic">O</Emphasis>-palmitoyl-1,2-<Emphasis Type="Italic">O</Emphasis>-isopropylidene-α-<Emphasis Type="SmallCaps">d</Emphasis>-glucofuranose in an organic solvent system by lipase-catalyzed esterification

In order to synthesize a sugar ester at high concentration, 1,2-O-isopropylidene-α-d-glucofuranose (IpGlc), which is one of the sugar acetals and is more hydrophobic than unmodified glucose, was esterified with palmitic acid at 40°C using immobilized lipase from Candida antarctica in some organic solvents or their mixtures. Acetone + t-butyl alcohol (3:1 v/v) improved both the initial reaction rate and yield after 80 h: the product reached its maximum value (240 mmol/kg solvent; ca. 110 g/kg solvent) when 400 mmol IpGlc/kg solvent and 1,200 mmol palmitic acid/kg solvent were used in this solvent mixture.     

δ-(<Emphasis Type="SmallCaps">l</Emphasis>-α-aminoadipyl)-<Emphasis Type="SmallCaps">l</Emphasis>-cysteinyl-<Emphasis Type="SmallCaps">d</Emphasis>-valine synthetase (ACVS): discovery and perspectives

The δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) tripeptide is the first dedicated intermediate in the biosynthetic pathway leading to the penicillin and cephalosporin classes of β-lactam natural products in bacteria and fungi. It is synthesized nonribosomally by the ACV synthetase (ACVS) enzyme, which has been purified and partially characterized from many sources. Due to its large size and instability, many details regarding the reaction mechanism of ACVS are still not fully understood. In this review we discuss the chronology and associated methodology that led to the discovery of ACVS, some of the main findings regarding its activities, and some recent/current studies being conducted on the enzyme. In addition, we conclude with perspectives on what can be done to increase our understating of this very important protein in the future.     

Cloning of the <Emphasis Type="Italic">Penicillium canescens</Emphasis> endo-1,4-β-glucanase gene <Emphasis Type="Italic">egl3</Emphasis> and the characterization of the recombinant enzyme

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-β-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for β-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54°C, respectively. The K _m and V _m values for CMC hydrolysis were determined; they amounted to 17.1 g/l and 0.31 μM/(mg s), respectively.     

cDNA Cloning and Functional Expression of the α-<Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin Frutalin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

cDNA clones encoding frutalin, the α-d-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa), were isolated and sequenced. The deduced amino acid sequences indicated that frutalin may be encoded by a family of genes. The NCBI database searches revealed that the frutalin sequence is highly homologous with jacalin and mornigaG sequences. Frutalin cDNA was re-amplified and cloned into the commercial expression vector pET-25b(+) for frutalin production in Escherichia coli. An experimental factorial design was employed to maximise the soluble expression of the recombinant lectin. The results indicated that temperature, time of induction, concentration of IPTG and the interaction between the concentration of IPTG and the time of induction had the most significant effects on the soluble expression level of recombinant frutalin. The optimal culture conditions were as follows: induction with 1 mM IPTG at 22°C for 20 h, yielding 16 mg/l of soluble recombinant frutalin. SDS-PAGE and Western blot analysis revealed that recombinant frutalin was successfully expressed by bacteria with the expected molecular weight (17 kDa). These analyses also showed that recombinant frutalin was mainly produced as insoluble protein. Recombinant frutalin produced by bacteria revealed agglutination properties and carbohydrate-binding specificity similar to the native breadfruit lectin.     

α-<Emphasis Type="SmallCaps">l</Emphasis>-Rhamnosidase of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> immobilized on ferromagnetic supports

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).     

Codon optimization,expression and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MA139 <Emphasis Type="Italic">β</Emphasis>-1,3-1,4-glucanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-1,3-1,4-Glucanase has been broadly used in feed and brewing industries. According to the codon bias of Pichia pastoris, the Bacillus subtilis MA139 β-1,3-1,4-glucanase gene was de novo synthesized and expressed in P. pastoris X-33 strain under the control of the alcohol oxidase 1 promoter. In a 10-L fermentor, the β-1,3-1,4-glucanase was overexpressed with a yield of 15,000 U/mL by methanol induction for 96 h. The recombinant β-1,3-1,4-glucanase exhibited optimal activity at 40°C and pH 6.4. The activity of the recombinant β-1,3-1,4-glucanase was not significantly affected by various metal ions and chemical reagents. To our knowledge, the expression of this β-1,3-1,4-glucanase from Bacillus sp. in P. pastoris is in relatively high level compared to previous reports. These biochemical characteristics suggest that the recombinant β-1,3-1,4-glucanase has a prospective application in feed and brewing industries.     

Syntheses of mucin-type <Emphasis Type="Italic">O</Emphasis>-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of <Emphasis Type="Italic">Bifidobacterium</Emphasis> endo-α-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminidase

Endo-α-N-acetylgalactosaminidase catalyzes the release of Galβ1-3GalNAc from the core 1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr) of mucin glycoproteins and synthetic p-nitrophenyl (pNP) α-linked substrates. Here, we report the enzymatic syntheses of core 1 disaccharide-containing glycopeptides using the transglycosylation activity of endo-α-N-acetylgalactosaminidase (EngBF) from Bifidobacterium longum. The enzyme directly transferred Galβ1-3GalNAc to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T and MUC1a when Galβ1-3GalNAcα1-pNP was used as a donor substrate. The enzyme was also found to catalyze the reverse-hydrolysis reaction. EngBF synthesized the core 1 disaccharide-containing oligosaccharides when the enzyme was incubated with either glucose or lactose and Galβ1-3GalNAc prepared from porcine gastric mucin using bifidobacterial cells expressing endo-α-N-acetylgalactosaminidase. Synthesized oligosaccharides are promising prebiotics for bifidobacteria.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-<Emphasis Type="Italic">Z</Emphasis>, <Emphasis Type="Italic">N</Emphasis>′-Formyl α-Amino Acid Derived Gem-Diamines

A variety of N-carbobenzoxy, N′-formyl gem-diaminoalkyl derivatives have been obtained through Goldsmith-Wick reaction of Z-α-amino acid/peptide acid derived isocyanates with 96% HCOOH in presence of 4-dimethylaminopyridine (DMAP) as catalyst. The reaction proceeds to completion within 2–4 h and results in good yields of the products isolated as stable solids.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.