Molecular Cloning of the Phytase Gene from Citrobacter braakii and its Expression in Saccharomyces cerevisiae

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppA_Ec and rAppA_Sc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppA_Sc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppA_Ec.     

Substrate specificity and membrane topologies of the iron-containing ω3 and ω6 desaturases from Mortierella alpina

Applied Microbiology and Biotechnology - Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of...     

γ-N-Methylasparagine in phycobiliproteins from the cyanobacteria Mastigocladus laminosus and Calothrix

Reinvestigation of the amino acid sequences of all phycobiliproteins from Mastigocladus laminosus showed that there is a post-translationally modified asparagine residue at position 72 of the phycobiliprotein subunits β^PC, β^AP and β^16.2. This residue was identified as γ-N-methylasparagine and it was also found in β^PE of Calothrix. This study also revealed some differences in the amino acid sequences β^AP and β^PC compared to the published data.     

Molecular Cloning and Stress-Dependent Expression of a Gene Encoding ω3-Fatty Acid Desaturase in the Microalga Dunaliella salina

A fragment of the gene des3-1 encoding 3 fatty acid desaturase was cloned from a cDNA library of the unicellular green galophilic alga Dunaliella salina. The comparative phylogenetic analysis of 3-desaturase amino acid sequences from diverse organisms placed the desaturase of D. salina between cyanobacteria and higher plants in the evolutionary range of desaturases. The expression of des3-1 was studied in D. salina cells exposed to low temperatures, high irradiance, and high CO₂ concentrations. Lowering the external temperature from 32 to 22°C produced a transient increase in the level of specific mRNA. Considerable accumulation of mRNA for 3-desaturase was also observed when CO₂ concentration in gas–air mixture was raised from 2 to 10%. An irradiation increase from 70 to 500 mol/(m² s) did not affect the level of specific mRNA. The latter evidence presumes that in Dunaliella cells, this desaturase is probably located in the endoplasmic reticulum, rather than in the chloroplast.     

α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

Characteristics of Trichoderma reesei β-xylosidase and its use in the hydrolysis of solubilized xylans

Summary The -xylosidase (EC 3.2.1.37) of Trichoderma reesei was purified and its characteristics and use in the hydrolysis of steamed birch xylan were studied. The enzyme was a glycoprotein with a molecular weight of 100000 as determined by SDS-gel electrophoresis and its isoelectric point was 4.7. The pH optimum was 4.0 and temperature optimum 60°C. -Xylosidase was competitively inhibited by xylose and the inhibition constant was 2.3 mM. The purified enzyme also showed -arabinofuranosidase activity.     

Isolation and expression of CsFAD7 and CsFAD8, two genes encoding ω-3 fatty acid desaturase from Camellia sinensis

The plastidial ω-3 fatty acid desaturase catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts and plays an important role in plant responses to environmental stress. In this study, the full-length cDNAs encoding two plastidial ω-3 desaturases, designated CsFAD7 and CsFAD8 (GenBank Accession No. JX943516 and KC847167, respectively), were isolated from the tea plant (Camellia sinensis L.) using RT-PCR and RACE. Codon usage analysis revealed that U- and A-ended codons were preferentially used in these two genes. Sequence analysis showed that the deduced amino acid sequences of CsFAD7 and CsFAD8 had high homology to plastidial ω-3 desaturases from other plant species. Expression analysis by real-time PCR revealed that both genes are tissue-specific and expressed the highest levels in shoots. Meanwhile, CsFAD7 and CsFAD8 responded to various abiotic stresses and hormones, but in very different manners. Taken together, these results suggest that CsFAD7 and CsFAD8 are both responsive to abiotic stress signals; however, they may play very different roles during stress tolerance in tea plants.     

Structure,chromosomal location and expression of a rice gene encoding the microsome ω-3 fatty acid desaturase

The -3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. A cDNA encoding the microsome -3 fatty acid desaturase (OsFAD3) and the corresponding genomic clone were isolated from rice (Oryza sativa L.). The OsFAD3 gene was composed of 8 exons and 7 introns. A microsatellite was present in the second exon of the OsFAD3 gene, showing polymorphism between Indica and Japonica rice varieties. The mapping of this microsatellite showed that the OsFAD3 gene was located on chromosome 11. Expression of the OsFAD3 cDNA in tobacco hairy root tissues and subsequent analysis of fatty acid compositions demonstrated the activity of the microsome -3 fatty acid desaturase. The OsFAD3 mRNA was abundant in root tissues, but was hardly detectable in leaves. In root tissues, a high level of the OsFAD3 mRNA was observed at 15 °C and 20 °C, with its level decreasing markedly at temperatures below 10 °C. The accumulation of the OsFAD3 mRNA in leaf tissues remained at quite low levels, both at normal growth temperatures and at chilling temperatures. Similar temperature responses of the OsFAD3 gene were observed both in chilling- tolerant and in chilling-intolerant rice cultivars.     

Fractionation of a commercial cellulase preparation from Penicillium funiculosum and its use in the purification of a water soluble α-glucan from milled barley

A commercial enzyme preparation from a selected strain of Penicillium funiculosum has been partially purified using a single stage chromatofocusing fractionation to produce an amylase-free mixture of hydrolytic enzymes. This mixture has been used to remove the non-starch polysaccharides from aqueous extracts of laboratory milled barley. The structure of the resulting purified α-glucan fraction has been examined by gel filtration before and after enzymic debranching and by iodine staining. The mild damage caused to the starch within the barley kernel releases a low molecular weight amylopectin molecule with no detectable amylose in the product. In this respect the product is different from that produced after severe, direct grinding of the purified barley starch where some amylose appears in the water soluble extract. Although the small amount of α-glucan is not of any quantitative industrial significance in itself, it does reflect the extent and type of physical damage which is taking place in the starch granule. The model proposed to explain these results - a starch granule with a solid amylose/amylopectin core but with a number of patches of protruding amylopectin clusters - may have important implications in an industrial context.     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Functional complementation of a periila ω3 fatty acid desaturase under the seed-specific SeFAD2 promoter

Functional characterization of the fatty acid desaturase genes and seed-specific promoters is prerequisite for altering the unsaturated fatty acid content of oilseeds by genetic manipulation. The ω-6 fatty acid desaturase (FAD2) and ω-3 fatty acid desaturase (FAD3) catalyze extra-plastidial desaturation of oleic acid to linoleic acid and linoleic acid to linolenic acid, respectively. These are major constituents in seed storage oils. Here, we report the complementation of a perilla linoleic acid desaturase (PrFAD3) cDNA under the seed-specific sesame FAD2 (SeFAD2) promoter in the Arabidopsis fad3 mutant. PrFAD3 is functionally active and the SeFAD2 promoter is applicable for modifying fatty acid composition in developing seeds. Transient expression of the GUS gene under that promoter in the developing seeds and leaves of sesame, soybean, and corn via microprojectile bombardment indicated that the SeFAD2 promoter likely will be useful for altering the seed phenotypes of dicot and monocot crops.     

Association of the FADS2 Gene with ω-6 and ω-3 PUFA Concentration in the Egg Yolk of Japanese Quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low ω-6:ω-3 PUFA ratio (h2 = 0.36–0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the ω-6 and ω-3 PUFAs (P < 0.05). No effects of the other SNPs were found—indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to ω-6 and ω-3 PUFA concentration in the egg yolk.