Characterization of hydroxyl groups of highly crystalline β-chitin under static tension detected by FT-IR

The different behavior of the OH groups of a highly crystalline β-chitin from Lamellibrachia sp. was revealed by FT-IR spectroscopy with static tension applied in the direction of the chain. Both OH groups shifted to higher wavenumbers under the tension, possibly because the force constant of the OH vibration intensified due to the erosion of hydrogen bonds, but the shift in the OH peak occurring at 3435 cm⁻¹ was 14% greater than that of the peak at 3470 cm⁻¹. The band at 3435 cm⁻¹ was assigned to O(3)H and that at 3470 cm⁻¹ to O(6)H, because the intramolecular hydrogen bond of O(3)H?O(5) occurs along the chitin chain's backbone and is likely to be more affected under tensile stress as a load carrier, than O(6)H?O(7)C, which is an intermolecular hydrogen bond located in a branch of the chain. Computation of an IR spectrum under stretching was conducted and supported the assignment of the two OH groups.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi

Soil isolates of mesophilic Penicillium monoverticillium CFR 2, Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 were cultivated in solid state fermentation (SSF) using wheat bran solid medium supplemented with α-chitin in order to produce chitinolytic enzyme. Under SSF cultivation, maximum enzymes (U/g IDS) production was 41.0 (endo-chitinase) and 195.4 (β-N-acetylhexosaminidase) by P. monoverticillium, 26.8 (endo-chitinase) and 222.1 (β-N-acetylhexosaminidase) by A. flavus and 13.3 (endo-chitinase) and 168.3 (β-N-acetylhexosaminidase) by F. oxysporum after 166?h of incubation. The crude endo-chitinase and β-N-acetylhexosaminidase derived from A. flavus and F. oxysporum revealed optimum temperature at 62?±?1°C, but the enzymes from P. monoverticillium showed optimum temperature at 52?±?1°C for maximum activity. Several fold increase in endo-chitinase and β-N-acetylhexosaminidase activities in the crude enzymes preparation was achieved after concentrating with polyethylene glycol. The concentrated crude chitinases from P. monoverticillium, A. flavus and F. oxysporum, respectively yielded 95.6, 96.6 and 96.1?mmol/l of N-acetyl-D: -glucosamine (GlcNAc) in 48?h of reaction from colloidal chitin. While, the crude enzyme preparations of P. monoverticillium, A. flavus and F. oxysporum produced 10.11, 6.85 and 10.7?mmol/l of GlcNAc respectively, in 48?h of reaction from crystalline α-chitin. HPLC analysis of colloidal chitin hydrolysates prepared with crude chitinases derived from P. monoverticillium, A. flavus and F. oxysporum revealed that the major reaction product was monomeric GlcNAc (~80%) and a small amount of (GlcNAc)(4) (~20%), indicating the potential of these enzymes for efficient production of GlcNAc from α-chitin.     

Purification and characterisation of a novel alkalophilic α-D-mannosidase from Pseudomonas fluorescens

An extracellular alkaline α-D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30°C. The K_m and V_max values of the purified enzyme towards p-nitrophenyl-α-D-mannopyranoside were determined to be 77 µM and 0.23 µM min^?1mg^?1 of protein, respectively. The α-D-mannosidase showed higher substrate specificity to α-1,3-mannobiose than other isomeric substrates such as α-1,2- and α-1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II α-mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic α-1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.     

Expression and biochemical characterisation of recombinant AceA, a bacterial α-mannosyltransferase

Biosynthesis of repeat-unit polysaccharides and N-linked glycans proceeds by sequential transfer of sugars from the appropriate sugar donor to an activated lipid carrier. The transfer of each sugar is catalysed by a specific glycosyltransferase. The molecular basis of the specificity of sugar addition is not yet well understood, mainly because of the difficulty of isolating these proteins. In this study, the aceA gene product expressed by Acetobacter xylinum, which is involved in the biosynthesis of the exopolysaccharide acetan, was overproduced in Escherichia coli and its function was characterised. The aceA ORF was subcloned into the expression vector pET29 in frame with the S·tag epitope. The recombinant protein was identified, and culture conditions were optimised for production of the soluble protein. The results of test reactions showed that AceA is able to transfer one α-mannose residue from GDP-mannose to cellobiose-P-P-lipid to produce α-mannose-cellobiose-P-P-lipid. AceA was not able to use free cellobiose as a substrate, indicating that the pyrophosphate-lipid moiety is needed for enzymatic activity. Received: 11 February 1999 / Accepted: 6 April 1999     

The isolation and characterisation of gas-cylinder membranes and α-granules fromAnabaena flos-aquae D 124

Summary Gas-cylinder membranes and -granules were isolated from cells of the blue-green alga,Anabaena flos-aquae D 124. Gas-cylinder membranes, which are 1,100 Å wide after isolation, show striations having a periodicity of 50 Å. The membranes appear globular in section with a spacing of about 40 Å between globules. There are strong indications that the globules are proteinaceous with molecular weights of 22,000±2,000. Possible homologies of these membranes with viral coat protein are discussed. -granules were shown by a variety of techniques to be polysaccharide.     

Secretion, purification, and characterisation of barley α-amylase produced by heterologous gene expression in Aspergillus niger

Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence. Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997     

Synthesis, characterization and cytocompatibility studies of α-chitin hydrogel/nano hydroxyapatite composite scaffolds

α-chitin hydrogel/nano hydroxyapatite (nHAp) composite scaffold have been synthesized by freeze-drying approach with nHAp and α-chitin hydrogel. The prepared nHAp and nanocomposite scaffolds were characterized using DLS, SEM, FT-IR, XRD and TGA studies. The porosity, swelling, degradation, protein adsorption and biomineralization (calcification) of the prepared nanocomposite scaffolds were evaluated. Cell viability, attachment and proliferation were investigated using MG 63, Vero, NIH 3T3 and nHDF cells to confirm that the nanocomposite scaffolds were cytocompatible and cells were found to attach and spread on the scaffolds. All the results suggested that these scaffolds can be used for bone and wound tissue engineering.     

Effects of galactomannan as carbon source on production of α-galactosidase by Thermomyces lanuginosus: Fermentation,purification and partial characterisation

Thermophilic fungus Thermomyces lanuginosus CBS 395.62/b strain is able to grow and synthesise extracellular α-galactosidase in media containing galactomannan such as locust bean gum (LBG) or guar gum (GG). Production of extracellular α-galactosidase was enhanced from 1.2 U/mL to 4–6 U/mL meaning about 3–5 times increase by optimisation of medium composition. This enzyme was purified to homogeneity by partial precipitation with 2-propanol and different liquid chromatographical steps. The developed purification protocol yielded 22% of enzyme activity with 900 purified fold. Molecular mass of the purified α-galactosidase enzyme was estimated to be 53 kDa. Maximal catalytic activity of the enzyme was obtained in the acidic pH range between pH 4.6 and 4.8 and in the temperature range 60–66 °C. More than 95% of enzyme activity was remaining after 1-day incubation at 70 °C and on pH in the range from 4.0 to 7.0. The enzyme activity was significantly stimulated by Mg²⁺, Mn²⁺ and K⁺ ions, while considerably inhibited by the presence of Ca²⁺, Ag⁺ and Hg²⁺.     

Isolation and partial characterisation of α-amylase components evolved during early wheat germination

The development of α-amylase (EC 3.2.1.1) activity in wheat was followed during 4 days of germination. The enzyme was purified and separated by gel chromotography into two distinct entities (α-amylase I and α-amylase II), with different molecular weights and isoelectric points. α-Amylase I contained a much higher content of sugars than α-amylase II, which decreased as the germination proceeded. The time sequence analysis of the starch degradation pattern showed that on the 4th day of germination, 15% of the total activity was present in α-amylase I and the rest in a-amylase II. Similarly, differences in the relative rates of synthesis of their isoenzymes were observed. α-Amylase I was resolved on the 4th day of germination, only into 3 isoenzymes, whereas α-amylase II could separate into 4 isoenzymes. The enzyme activity was however maximal in the most electropositive isoenzyme in both the components.     

Self-assembly and mineralization of genetically modifiable biological nanofibers driven by β-structure formation

Bioinspired mineralization is an innovative approach to the fabrication of bone biomaterials mimicking the natural bone. Bone mineral hydroxylapatite (HAP) is preferentially oriented with c-axis parallel to collagen fibers in natural bone. However, such orientation control is not easy to achieve in artificial bone biomaterials. To overcome the lack of such orientation control, we fabricated a phage-HAP composite by genetically engineering M13 phage, a nontoxic bionanofiber, with two HAP-nucleating peptides derived from one of the noncollagenous proteins, Dentin Matrix Protein-1 (DMP1). The phage is a biological nanofiber that can be mass produced by infecting bacteria and is nontoxic to human beings. The resultant HAP-nucleating phages are able to self-assemble into bundles by forming β-structure between the peptides displayed on their side walls. The β-structure further promotes the oriented nucleation and growth of HAP crystals within the nanofibrous phage bundles with their c-axis preferentially parallel to the bundles. We proposed that the preferred orientation resulted from the stereochemical matching between the negatively charged amino acid residues within the β-structure and the positively charged calcium ions on the (001) plane of HAP crystals. The self-assembly and mineralization driven by the β-structure formation represent a new route for fabricating mineralized fibers that can serve as building blocks in forming bone repair biomaterials and mimic the basic structure of natural bones.     

Preparation,characterization, bioactive and cell attachment studies of α-chitin/gelatin composite membranes

The chitin/gelatin composite membranes were prepared by mixing of chitin hydrogel with gelatin. The prepared composite membranes were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical, swelling, enzymatic degradation and thermal studies. The XRD pattern of the chitin/gelatin composite membranes showed almost the same pattern as α-chitin. The bioactivity studies of these chitin/gelatin membranes were carried out with the simulated body fluid solution (SBF) for 7, 14 and 21 days followed by the characterization with the scanning electron microscopy (SEM) and Energy Dispersive Spectrum (EDS) studies. The SEM and EDS studies confirmed the formation of calcium phosphate layer on the surface of chitin/gelatin membranes. Biocompatibility of the chitin/gelatin membrane was assessed using human MG-63 osteoblast-like cells. After 48 h of incubation, it was found that the cells had attached and completely covered the membrane surface. Thus, the prepared chitin/gelatin membranes are bioactive and are suitable for cell adhesion suggesting that these membranes can be used for tissue-engineering applications.     

Screening of high α-arbutin producing strains and production of α-arbutin by fermentation

A mutant Xanthomonas maltophilia BT-112 with high α-anomer-selective glycosylation activity was screened by a series of mutation methods including UV light, N-methyl-N-nitro-N-nitroso-guanidine treatment and quick neutron mutation. The α-arbutin titer increased 15-folds compared with the parent strain. The optimal conditions for culture medium and the operational conditions for lab-scale fermenter were investigated. Under optimized conditions, the maximal hydroquinone (HQ) tolerance of cells and yield of α-arbutin were 120 mM and 30.6 g/l, respectively. The molar conversion yield of α-arbutin based on the amount of HQ supplied reached 93.6 %. The product was identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the results in this work provide a one-step and cost-effective method for the large-scale production of α-arbutin.     

Overproduction and secretion of α-ketoglutaric acid by microorganisms

This mini-review presents a summary of research results of biotechnological production of alpha-ketoglutaric acid (KGA) by bacteria and yeasts. KGA is of particular industrial interest due to its broad application scope, e.g., as building block chemical for the chemical synthesis of heterocycles, dietary supplement, component of infusion solutions and wound healing compounds, or as main component of new elastomers with a wide range of interesting mechanical and chemical properties. Currently KGA is produced via different chemical pathways, which have a lot of disadvantages. As an alternative several bacteria and yeasts have already been studied for their ability to produce KGA as well as for conditions of overproduction and secretion of this intermediate of the tricarboxylic acid cycle. The aim of this mini-review was to summarize the known data and to discuss the potentials of biotechnological processes of KGA production.     

Substrate-selective and calcium-independent activation of CaMKII by α-actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Purification, characterisation and mutagenic enhancement of a thermoactive α-amylase from Bacillus subtilis

Bacillus subtilis was isolated from flour mill wastes. It produced a thermostable α-amylase in complex media containing starch. Amylase activity was optimal at the exponential phase and was more strongly expressed with sorghum, yam peel and corn starch than soluble potato starch. The enzyme was purified 24-fold to a specific activity of 2200 U mg⁻¹, with a yield of 10%. It yielded a single band when subjected to SDS-PAGE and an apparent molecular mass of 54780 was determined by mass spectrometry. The enzyme, which was optimally active at 80°C and pH 5.6, released saccharides with a polymerisation degree of 1–6 following hydrolysis of yam peel, sorghum and corn starch. Cells of B. subtilis were exposed to ultraviolet irradiation and N-methyl-N′-nitro-N-nitrosoguanidine. Hyperproductive mutants were obtained by these treatments. Received 14 February 1997/ Accepted in revised form 13 August 1997