Sequence analysis of cyclodextrin glycosyltransferase from the alkaliphilic Bacillus agaradhaerens

The gene encoding an alkaline active cyclodextrin glycosyltransferase (CGTase) from the alkaliphilic B. agaradhaerens LS-3C was cloned and sequenced. It encodes a mature polypeptide of 679 amino acids with a molecular mass of 76488 Da. The deduced amino acid sequence of the mature CGTase revealed 99 and 95% identity to the CGTase sequences from the other B. agaradhaerens strains, DSM 8721^T and 9948, respectively. The next closest identity was of 59% with B. clarkii enzyme. CGTases from B. agaradhaerens, B. clarkii, and B. firmus/lentus formed a phylogenetically separated cluster from the other CGTases of Bacillus spp. origin. A number of usually conserved residues in the CGTases were found to be replaced in the sequence of B. agaradhaerens enzyme. The sequence analysis indicated the enzyme to be close to the so-called `intermediary enzymes' in the -amylase family.     

Production of 2-O-α-d-glucopyranosyl l-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp.

Transglycosylation to produce a 2-O--d-glucopyranosyl l-ascorbic acid (AA-2G) was studied using cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. A series of maltooligosaccharides substituted 2-O-derivatives of l-ascorbic acid (AA) were analyzed by HPLC. The maltooligosaccharides were hydrolyzed by glucoamylase to give AA-2G. CGTase also produced AA-2G using dextrin as a glycosyl donor and AA as an acceptor. CGTase utilized -, -, and -CDs, amylose, soluble starch and corn starch as glycosyl donors but not glucose.     

Production of 22:2<Superscript>Δ5,Δ13</Superscript> and 20:1<Superscript>Δ5</Superscript> in <Emphasis Type="Italic">Brassica carinata</Emphasis> and soybean breeding lines via introduction of <Emphasis Type="Italic">Limnanthes</Emphasis> genes

Seed oils of meadowfoam (Limnanthes douglasii, L. alba) contain very long-chain fatty acids of strategic importance for a number of industrial applications. These include the monoene 20 1⁵ and the diene 22:2^5,13. Engineering of meadowfoam-type oils in other oilseed crops is desirable for the production of these fatty acids as industrial feedstocks. Accordingly, we have targeted Brassica carinata and soybean (Glycine max) to trangenically engineer the biosynthesis of these unusual fatty acids. An L. douglasii seed-specific cDNA (designated Lim Des5) encoding a homolog of acyl-coenzyme A desaturases found in animals, fungi and cyanobacteria was expressed in B. carinata, which resulted in the accumulation of up to 10% 22:2^5,13 in the seed oil. In soybean, co-expression of Lim Des5 with a cDNA (Lim FAE1) encoding an FAEl (elongase complex condensing enzyme) homolog from L. douglasii resulted in the accumulation of 20:1⁵ to approximately 10% of the total fatty acids of seeds. The content of C₂₀ and C₂₂ fatty acids was also increased from <0.5% in non-transformed soybean seeds to >25% in seeds co-expressing the Lim. douglasii Des5 and FAE1 cDNAs. In contrast, expression of the Lim Des5 in Arabidopsis did not produce the expected 20:2^5,11 in the seed oil. Cumulatively, these results demonstrate the utility of soybean and B. carinata for the production of vegetable oils containing novel C₂₀ and C₂₂ fatty acids, and confirm that the preferred substrates of the Lim Des5 are 20:0 and 22:1³, respectively.     

Cloning, analysis and expression of the gene for β-glycosidase of Thermus caldophilus GK24 and properties of the enzyme

The gene (bglT) encoding Thermus caldophilus GK24 -glycosidase (Tca -glycosidase) was cloned and sequenced. The gene contains an open reading frame encoding 431 amino acids with a M _r of 48 658 Da. The bglT gene was expressed under the control of tac promoter on a high-copy plasmid in E. coli. The recombinant Tca -glycosidase was purified 41.5-fold with a 59% yield and a specific activity of 83 U mg^–1 protein.     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Enhancement of production of cloned α-amylase by lactic acid feeding from recombinant Saccharomyces cerevisiae using a SUC2 promoter

-Amylase production was higher (13 units ml^–1) when a recombinant Saccharomyces cerevisiae containing a SUC2 promoter was grown with 10 g lactic acid l^–1 than without addition (8 units ml^–1). With continuous lactic acid feeding in the inducing phase, -amylase increased to 79 units ml^–1 in a 1-l jar fermenter.     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

Tepidimonas taiwanensis sp. nov., a novel alkaline-protease-producing bacterium isolated from a hot spring

The bacterial strain designated I1-1^T was isolated from a hot spring located in the Pingtung area, southern Taiwan. Cells of this organism were Gram reaction negative rods, motile by a single polar flagellum. Optimum conditions for growth were 55°C and pH 7. Strain I1-1^T grew well in lower nutrient media such as 5–10% Luria–Bertani broth, and its extracellular products expressed alkaline protease activity. The 16S rRNA gene sequence analysis indicates that strain I1-1^T is a member of -Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA–DNA similarity data, whole-cell protein analysis, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Tepidimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain I1-1^T were 16:0 (about 41%), 18:1 7c (about 13%), and summed feature 3 [16:1 7c or 15:0 iso 2OH or both (about 26%)]. Its DNA base ratio was 68.1 mol%. We propose to classify strain I1-1^T (=BCRC 17406^T=LMG 22826^T) as Tepidimonas taiwanensis sp. nov.     

Synthesis of β(1–2)glucan in Rhizobium loti

Fast growing strains of Rhizobium loti isolated from nodules of Lotus tenuis of the flooding Pampas of Argentina produced cellular (1–2)glucans having a higher degree of polymerization and more anionic substituents than (1–2)glucans accumulated by Agrobacterium tumefaciens cells. Inner membranes of R. loti contained a 235 kDa (1–2)glucan intermediate protein indistinguishable by polyacrylamide gel electrophoresis from the intermediate protein present in A. tumefaciens inner membranes. Incubation of inner membrannes of R. loti with UDP-Gle led to the formation of neutral (1–2)glucans with a higher degree of polymerization than glucans formed by A. tumefaciens inner membranes.Introduction in R. loti strains of plasmid pCD523 containing A. tumefaciens chvA and chvB virulence regions yielded strains that accumulated 4 times more cellular (1–2)glucans than wild type cells. This glucan was, regarding anionic substitution and degree of polymerization, indistinguishable from A. tumefaciens (1–2)glucans. Furthermore inner membranes of these R. loti exoconjugant cells contained higher levels of the 235 kDa (1–2)glucan intermediate protein and formed in vitro 8 times more neutral (1–2)glucan with a degree of polymerization corresponding to A. tumefaciens (1–2)glucan than inner membranes isolated from wild type cells.It was concluded that A. tumefaciens chvB gene is expressed in R. loti and determined the degree of polymerization of (1–2)glucan.Abbreviations Nod⁺ effective nodulation - Vir⁺ virulent - Vir^- avirulent - Trp^r trimethoprim resistence - Tc^r tetracycline resistence - TCA trichloroacetic acid - PMSF phenyl methyl sulfonyl fluoride     

Molecular cloning and sequencing analysis of a β-tubulin gene from Lupinus albus

Genomic -Dash library constructed from Lupinus albus nuclear DNA was screened using a fragment of the -tubulin cDNA ( 8–31) clone of Chlamydomonas reinhardtii as probe. One of the positive recombinant phages was isolated, subcloned and analysed by sequencing. We present here nucleotide and derived amino acid sequences of the -tubulin gene, designated as L1 and identified by similarity with other -tubulins. The L1-encoded protein reveals a very high degree of similarity with other plant tubulins and contains consensus sequences for binding guanine base, phosphate and Mg²⁺. Northern analysis of total RNA isolated from roots, leaves, flowers and pools revealed that Lupinus albus -tubulin genes are constitutively expressed in all studied plant tissues.     

Variation in stable isotope signatures of seston and a zooplanktivorous fish in a eutrophic Chinese lake

Temporal and spatial changes in ¹³C and ¹⁵N of seston (mainly phytoplankton) and isotopic relationship between seston and the lake anchovy (Coilia ectenes) were studied in the large eutrophic freshwater Lake Chaohu in China. Much of the spatial and temporal variation in ¹³C of lake anchovies was explained by variation in seston, indicating a strong link between pelagic primary production and higher order consumers. Because the lake is shallow, there were no significant differences in ¹³C and ¹⁵N of seston between surface and overlying waters. Spatially, the relatively high ¹³C and ¹⁵N of seston in the western part of the lake might be due to high levels of anthropogenically derived N and C introduced from the surrounding cities through sewage drainage systems. The trophic position of the lake anchovy in the food web of Lake Chaohu was estimated to be 2.9–4.1 (3.5 ± 0.4), which agrees well with the previous stomach content analysis suggesting that the lake anchovy fed both on zooplankton and small planktivorous fishes.     

Functional characterisation of two cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>-fusion desaturases from<Emphasis Type="Italic"> Anemone leveillei</Emphasis>: the unexpected identification of a fatty acid Δ<Superscript>6</Superscript>-desaturase

The Ranunculaceae are known to accumulate a wide range of unusual fatty acids in their seed lipids, and this variability has been advocated as a taxonomic marker. The Anemone species, Anemone leveillei L. and Anemone rivularis Buch.-Ham., have previously been reported to accumulate ⁵-desaturated fatty acids in their seed tissue [K. Aitzetmüller (1995) Plant Syst Evol 9:229–240]. Two cDNAs, AL1 and AL2, with similarity to plant cytochrome b₅-fusion "front-end" desaturases were isolated from developing seeds of A. leveillei and their function identified by expression in Saccharomyces cerevisiae. AL2 was characterised as a sphingolipid long-chain-base ⁸-desaturase, while AL1 acted as a fatty acid desaturase. However, AL1 did not produce ⁵-desaturated fatty acids as expected; instead, when expressed in transgenic S. cerevisiae or Arabidopsis thaliana this enzyme was functionally characterised as a ⁶-desaturase. Northern analysis confirmed the expression of this gene in seed tissue and leaf tissue of A. leveillei, though ⁶-desaturated fatty acids were found to accumulate only in the leaf tissue. The unexpected characterisation of a ⁶-desaturase in A. leveillei has implications for the use of fatty acids in chemotaxonomic studies. This is also the first report of a higher-plant ⁶-desaturase from a family other than the Boraginaceae.Abbreviations ALA -linolenic acid - DMOX 4,4-dimethyloxazoline - EDA eicosadienoic acid - FAME fatty acid methyl ester - GLA -linolenic acid - LA linoleic acid - LCB long chain base - ORF open reading frame - OTA octadecatetraenoic acid     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

Mannosyl-β(1-4)-N-acetylglucosaminyl-β(1-N)-urea,a compound isolated from the urine of patients with β-mannosidosis

A previously unknown substance, mannosyl-(1–4)-N-acetylglucosaminyl-(1-N)-urea, has been isolated from the urine of patients with -mannosidosis in addition to the main metabolite mannosyl-(1–4)-N-acetylglucosamine. Structural investigation was carried out by fast atom bombardment mass spectrometry and high-resolution¹H-nuclear magnetic resonance spectroscopy at 500 MHz. It was postulated that the occurrence of this carbohydrate-urea conjugate in urine results mainly from urine handling.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Kinetics of beta-glucosidase production by Saccharomyces cerevisiae recombinants harboring heterologous bgl genes

The maximum productivity of -glucosidase by Saccharomyces cerevisiaerecombinants under the control of GALI promoter was 100 IU l^–1 h^–1. The highest productivity of -glucosidase by a S. cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g^–1 cellobiose and g ethanol l^–1 h^–1, respectively.     

Symmetric interspecies hybrids of mouse and human hemoglobin: Molecular basis of their abnormal oxygen affinity

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [ ₂ ^M ₂ ^H and ₂ ^H ₂ ^M (H=human, M=mouse)], representing 19 and 27 sequence differences per dimers (as compared with human dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O₂ binding. The intrinsic O₂ affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid ₂ ^M ₂ ^H has high O₂ affinity (compared to either human or mouse Hb), while the interspecies hybrid ₂ ^H ₂ ^M exhibits a very low O₂ affinity. These results suggest that the mouse chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse and chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse chain. DPG binds the tetramer in the central cavity formed by the two subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the chain present in the tetramer. The mouse chain reduces considerably the DPG right shift of the O₂ affinity of the human-chain containing hybrid. Sequence analysis suggest that perturbations of the _{1 1} (not the _{1 2}) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse chains in transgenic mouse expressing human ^S chains.