The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and (15)N uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C(34)-C(55) disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF-Pm3 binds to LPS, lipid A and to OM-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM-174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF-Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A-binding site of ALF-Pm3. Such a binding site, located on the ALF-Pm3 beta-sheet and involving seven charged residues, is well conserved in ALF-L from Limulus polyphemus and in ALF-T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that beta-hairpin synthetic peptides corresponding to ALF-L beta-sheet bind to LPS. Delineating lipid A-binding site of ALFs will help go further in the de novo design of new antibacterial or LPS-neutralizing drugs.
Candida molischiana 35M5N β-glucosidase was immobilized to
Duolite A-568 resin. Higher
immobilization efficiency (86%) was achieved with citrate−phosphate
buffer (0.1 M) at pH 4. The
study of the immobilized β-glucosidase demonstrated that the
physicochemical properties were
similar to those of the free enzyme. Free and immobilized
β-glucosidase were used to treat muscat
wine and apricot fruit juice. GC−MS analysis indicated a
significant increase in the flavor
compounds nerol, geraniol, linalool, 2-phenylethanol, and benzyl
alcohol in the muscat wine and
linalool, α- and γ-terpinene, α-terpineol, 2-phenylethanol, and
α-pinene in the apricot fruit juice.
The immobilized β-glucosidase was found to be very stable under
fruit juice or wine conditions and
could be used repeatedly for several hydrolyses of bound aroma.
The efficiency of this experimental
catalyst was successfully tested with several fruit juices and wines
containing various amounts of
precursors.
Keywords: Wines; fruit juices; aroma precursors; β-glucosidase;
immobilization
The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin‐containing medium, was found to produce an intracellular β‐glucosidase. The enzyme was purified by chromatofocusing, ion‐exchange chromatography and gel filtration. The molecular mass of the purified intracellular β‐glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β‐glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β‐glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)‐β, (1→4)‐α and (1→6)‐α linkage configuration. From Lineweaver–Burk plots, Km values of 0·07 mmol l−1 and 3·7 mmol l−1 were found for p‐nitrophenyl‐β‐D‐glucopyranoside and linamarin, respectively. The β‐glucosidase was competitively inhibited by glucose and by D‐gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β‐glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.
The β‐glucosidase of Hanseniaspora vineae was purified by ion‐exchange chromatography and gel filtration. Its molecular weight was 295000 PT 15000, its optimum pH was between 6 and 6–5, and its optimum temperature was 55°C. The enzyme was active against different soluble glucosides with β(1–2), β(1–3), β(1–4), β(1–6) and even aP(1–4) configurations. A glucosyltransferase activity appeared in the presence of ethanol. The enzyme was constitutive but its synthesis was repressed by glucose.
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