<Emphasis Type="Italic">Sphingomonas rosea</Emphasis> sp. nov. and <Emphasis Type="Italic">Sphingomonas swuensis</Emphasis> sp. nov., rosy colored <Emphasis Type="Italic">β</Emphasis>-glucosidase-producing bacteria isolated from soil

Two strains PB196^T and PB62^T of Gram-negative, non-motile, and non-spore-forming bacteria, were isolated from soil in South Korea and characterized to determine their taxonomic positions. 16S rRNA gene sequence analysis showed that the two strains belonged to the genus Sphingomonas. The highest degree of sequence similarity of strain PB196^T was found with PB62^T (98.9%), Sphingomonas humi PB323^T (98.9%), Sphingomonas kaistensis PB56^T (98.2%), and Sphingomonas astaxanthinifaciens TDMA-17^T (98.0%). The highest degree of sequence similarity of strain PB62^T was found with Sphingomonas humi PB323^T (98.8%), Sphingomonas astaxanthinifaciens TDMA-17^T (98.2%), and Sphingomonas kaistensis PB56^T (98.1%). Chemotaxonomic data revealed that they possessed ubiquinone-10 (Q-10) as common in the genus Sphingomonas, that the predominant fatty acids were summed feature 7 (C_18:1 ω7c/ω9t/ω12t), summed feature 4 (C_16:1 ω7c/C_15:0 iso 2OH), C_16:0, and C_17:1 ω6c, and that they contained sphingoglycolipid, phosphatidylglycerol (PG), and phosphatidyle-thanolamine (PE) in common but they showed difference for diphosphatidylglycerol (DPG). Based on these data, PB196^T (=KCTC 12339^T =JCM 16604^T) and PB62^T (=KCTC 12336^T =JCM 16605^T =KEMB 9004-005^T) should be classified as type strains of two novel species, for which the names Sphingomonas rosea sp. nov. and Sphingomonas swuensis sp. nov. are proposed, respectively.     

Cloning,expression, and characterization of <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Exiguobacterium</Emphasis> sp. DAU5 and transglycosylation activity

A gram-negative bacterium, designated strain DAU5, was isolated from shrimp shell samples because it demonstrated high β-glucosidase activity. Through 16S rDNA gene sequence analysis the strain was identified as belonging to the genus Exiguobacterium. The β-glucosidase gene of Exiguobacterium sp. DAU5 was successfully cloned by the shotgun method. Nucleotide sequence determination by sodium dodecyl sulfate-ployacrylamide gel electrophoresis indicated that the gene for the enzyme contained 1,350 bp, was coded by 450 amino acids, and was 52 kDa. The polypeptide exhibits significant homology with other bacterial β-glucosidases and belongs to the Glycoside Hydrolase Family 1. The β-glucosidase was purified by a His-fusion purification system. The optimal pH and temperature of the enzyme were 7.0 and 45°C, respectively. The enzyme activity was strongly inhibited by Ca²⁺, and Li⁺, K⁺, Zn²⁺, Mg²⁺, Na²⁺, Ni²⁺, and EDTA partially inhibited the enzyme activity. The BglA showed the highest activity with p-NPG and MUG. However, strain DAU5 β-glucosidase, which is for degradation of oligosaccharides, is expected to be useful for the fermentation of cellulose degradation and the transglycosylation of saccharides.     

Biochemical characterization of <Emphasis Type="Italic">Candida albicans</Emphasis> α-glucosidase I heterologously expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, α-glucosidase I removes the outermost α1,2-glucose unit from the N-linked core Glc₃Man₉GlcNAc₂. We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3′-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed α-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.     

Hydrolysis of soy isoflavone glycosides by recombinant β-glucosidase from hyperthermophile <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

A recombinant Thermotoga maritima β-glucosidase A (BglA) was purified to homogeneity for performing enzymatic hydrolysis of isoflavone glycosides from soy flour. The kinetic properties K _m, k _cat, and k _cat/K _m of BglA towards isoflavone glycosides, determined using high-performance liquid chromatography, confirmed the higher efficiency of BglA in hydrolyzing malonylglycosides than non-conjugated glycosides (daidzin and genistin). During hydrolysis of soy flour by BglA at 80°C, the isoflavone glycosides (soluble form) were extracted from soy flour (solid state) into the solution (liquid state) in thermal condition and converted to their aglycones (insoluble form), which mostly existed in the pellet to be separated from BglA in the reaction solution. The enzymatic hydrolysis in one-step and two-step approaches yielded 0.38 and 0.35 mg genistein and daidzein per gram of soy flour, respectively. The optimum conditions for conversion of isoflavone aglycones were 100 U per gram of soy flour, substrate concentration 25% (w/v), and incubation time 3 h for 80°C.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Biodegradation of sulfamethazine by an isolated thermophile–<Emphasis Type="Italic">Geobacillus</Emphasis> sp. S-07

Sulfamethazine (SM2) is an antimicrobial drug that is frequently detected in manure compost, is difficult to degrade at high temperatures and is potentially threatening to the environment. In this study, a thermophilic bacterium was isolated from the activated sludge of an antibiotics pharmaceutical factory; this bacterium has the ability to degrade SM2 at 70?°C, which is higher than the traditional manure composting temperature. The strain S-07 is closely related to Geobacillus thermoleovorans based on its 16S rRNA gene sequence. The optimal conditions for the degradation of SM2 are 70?°C, pH 6.0, 50 rpm rotation speed and 50 mL of culture volume. More than 95% of the SM2 contained in media was removed via co-metabolism within 24 h, which was a much higher percentage than that of the type strain of G. thermoleovorans. The supernatant from the S-07 culture grown in SM2-containing media showed slightly attenuated antibacterial activity. In addition, strain S-07 was able to degrade other sulfonamides, including sulfadiazine, sulfamethoxazole and sulfamerazine. These results imply that strain S-07 might be a new auxiliary bacterial resource for the biodegradation of sulfonamide residue in manure composting.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Synthesis of α-cyclopiazonic acid by fungi of the genus <Emphasis Type="Italic">Aspergillus</Emphasis>

The presence of α-cyclopiazonic acid has been studied among metabolites of Aspergillus fungi. The study was performed with 138 cultures of 13 species obtained from the All-Russia Collection of Microorganisms and the collection of our institute. α-Cyclopiazonic acid was most frequently encountered among the metabolites of the section Flavi (the ability to synthesize α-cyclopiazonic acid was expressed in 61% of the strains of A. flavus, 83% of the strains of A. oryzae, and all strains of A. tamarii). This expression index for A. versicolor was less than 5%. We showed for the first time that α-cyclopiazonic acid is produced by A. fumigatus and A. phoenicis (expression in 30% of the strains of either species).     

Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite <Emphasis Type="Italic">Neotermes koshunensis</Emphasis> in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Neotermes koshunensis is a lower termite that secretes endogenous β-glucosidase in the salivary glands. This β-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-β-d-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn²⁺ and glycerol. The K _m and V _max values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-β-d-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Overexpression of <Emphasis Type="Italic">ppc</Emphasis> or deletion of <Emphasis Type="Italic">mdh</Emphasis> for improving production of γ-aminobutyric acid in recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

l-Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced l-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, l-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3?±?3.4, 24.8?±?0.7, and 25.5?±?3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7?±?0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0?±?0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.     

Cloning,extracellular expression and characterization of a predominant β-CGTase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. G1 in <Emphasis Type="Italic">E. coli</Emphasis>

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of β-CD.     

Biochemical characterization of a raw starch degrading <Emphasis Type="Italic">α</Emphasis>-amylase from the Indonesian marine bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. ALSHL3

An Indonesian marine bacterial isolate, which belongs to genus of Bacillus sp. based on 16S rDNA analysis and was identified as Bacillus filicolonicus according to its morphology and physiology, produced a raw starch degrading α-amylase. The partially purified α-amylase using a maize starch affinity method exhibited an optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme retained 72% of its activity in the presence of 1.5 M NaCl. Scanning electron micrographs showed that the α-amylase was capable of degrading starch granules of rice and maize. This α-amylase from Bacillus sp. ALSHL3 was classified as a saccharifying enzyme since its major final degradation product was glucose, maltose, and maltotriose.     

Synthesis and transformations of a pyrazole containing <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-didehydro-<Emphasis Type="Italic">α</Emphasis>-amino acid derivatives

Summary.  2H-Pyran-2-ones 1 were transformed with various hydrazines into (E)- or (Z)-α,β-didehydro-α-amino acid (DDAA) derivatives 4 (and 7) containing a highly substituted pyrazolyl moiety attached at the β-position. With heterocyclic hydrazines, the products 4 were accompanied also by decarboxylated enamines E-6. In order to separate (E/Z)-mixtures of acids, they were transformed to the corresponding methyl esters 9 and 10 by the application of diazomethane. Catalytic hydrogenation under high pressures with Pd/C as a catalyst resulted in the formation of racemic alanine derivatives 11. Received January 29, 2002 Accepted May 27, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated with deep respect to Professor Waldemar Adam on the occasion of his 65^th birthday. Acknowledgements We thank the Ministry of Education, Science and Sport of the Republic of Slovenia for the financial support (P0-0503-103). Dr. B. Kralj and Dr. D. Žigon (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) are gratefully acknowledged for the mass measurements. Authors' address: Prof. Marijan Kočevar, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia, E-mail: marijan.kocevar@uni-lj.si     

Insoluble but enzymatically active <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.     

High-level expression,purification and characterization of a recombinant medium-temperature <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase (AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml⁻¹) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg⁻¹ having optimal activity at pH 6.0 and 60°C.     

Biological Characterisation of <Emphasis Type="Italic">Haliclona</Emphasis> (?<Emphasis Type="Italic">gellius</Emphasis>) sp.: Sponge and Associated Microorganisms

We have characterised the northern Pacific undescribed sponge Haliclona (?gellius) sp. based on rDNA of the sponge and its associated microorganisms. The sponge is closely related to Amphimedon queenslandica from the Great Barrier Reef as the near-complete 18S rDNA sequences of both sponges were identical. The microbial fingerprint of three specimens harvested at different times and of a transplanted specimen was compared to identify stably associated microorganisms. Most bacterial phyla were detected in each sample, but only a few bacterial species were determined to be stably associated with the sponge. A sponge-specific β- and γ-Proteobacterium were abundant clones and both of them were present in three of the four specimens analysed. In addition, a Planctomycete and a Crenarchaea were detected in all sponge individuals. Both were closely related to operational taxonomic units that have been found in other sponges, but not exclusively in sponges. Interestingly, also a number of clones that are closely related to intracellular symbionts from insects and amoeba were detected.