Production of pure β-glucan by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> after pullulan synthetase gene disruption

By disruption of the pullulan synthetase gene (pul) of Aureobasidium pullulans IMS822 KCTC11179BP, we constructed a mutant strain, A. pullulans NP1221, which produced a pure β-glucan exopolysaccharide. The mutant NP1221 was white, whereas the wild-type strain produced a black dye. When we compared fermentation kinetics between wide-type and mutant strains, the mutant NP1221 did not produce pullulan. Substrate uptake rate and β-glucan production were similar in both strains. However, the biomass yield of mutant NP1221 was 2.3-fold (9.2 g l⁻¹) greater than that of wild-type.     

Characterization of a family 54 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K _m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V _max of 34.8 μmol min⁻¹ mg protein⁻¹. Arabinose acted as a noncompetitive inhibitor with a K _i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Heterologous expression and secretion of <Emphasis Type="Italic">Lactobacillus amylovorus</Emphasis> α-amylase in <Emphasis Type="Italic">Leuconostoc citreum</Emphasis>

To develop a gene expression system for Leuconostoc genus, construction of expression vector and expression of a heterologus protein in Leuconostoc was performed. α-Amylase gene from Lactobacillus amylovorus was cloned into a Leuconostoc cloning vector, pLeuCM, with its own signal peptide. pLeuCMamy was introduced into Leuconostoc citreum CB2567 and a successful expression of α-amy gene was confirmed by enzyme activity assays. About 90% of α-amylase activity was detected in the culture broth, revealing most of expressed α-amylase was secreted out cells. The signal sequence of α-amy gene is a good candidate for the secretion of heterologous protein by using Leuconostoc host-vector system.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

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.     

Molecular cloning,overexpression and characterization of the raw-starch-digesting <Emphasis Type="Italic">α</Emphasis>-amylase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low K _m of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high K _i value (350 mM).     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

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     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

Cloning and characterization of a maltotriose-producing α-amylase gene from <Emphasis Type="Italic">Thermobifida fusca</Emphasis>

The gene (tfa), encoding a maltotriose-producing α-amylase from Thermobifida fusca NTU22, was cloned, sequenced and expressed in Escherichia coli. The gene consists of 1,815 base pairs and encodes a protein of 605 amino acids. The base composition of the tfa coding sequence is 69% G+C and the protein has a predicted pI value of 5.5. The deduced amino acid sequence of the tfa amylase exhibited a high degree of similarity with amylases from Thermomonospora curvata and Streptomyces amylases. The purified amylase could be detected as a single band of about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The properties of purified amylase from the E. coli transformant are similar to that of an amylase purified from the original T. fusca NTU22.     

Molecular cloning and characterization of <Emphasis Type="Italic">α</Emphasis>-amylase/subtilisin inhibitor from rhizome of <Emphasis Type="Italic">Ligusticum chuanxiong</Emphasis>

Objectives

To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine.

Results

The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin. In addition, the expression level of LASI in rhizome was higher than that in leaf and LASI expression was enhanced by salt, chilling and drought treatment.

Conclusions

This is the first member of the Kunitz-protease inhibitor family identified in traditional Chinese medicine and it might be involved in the plant defense responses against lepidopterous pests, microorganisms and abiotic stresses.

     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Optimization of ethanol,citric acid,and α-amylase production from date wastes by strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>, <Emphasis Type="Italic">Aspergillus niger</Emphasis>, and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The present study deals with submerged ethanol, citric acid, and α-amylase fermentation by Saccharomyces cerevisiae SDB, Aspergillus niger ANSS-B5, and Candida guilliermondii CGL-A10, using date wastes as the basal fermentation medium. The physical and chemical parameters influencing the production of these metabolites were optimized. As for the ethanol production, the optimum yield obtained was 136.00 ± 0.66 g/l under optimum conditions of an incubation period of 72 h, inoculum content of 4% (w/v), sugars concentration of 180.0 g/l, and ammonium phosphate concentration of 1.0 g/l. Concerning citric acid production, the cumulative effect of temperature (30°C), sugars concentration of 150.0 g/l, methanol concentration of 3.0%, initial pH of 3.5, ammonium nitrate concentration of 2.5 g/l, and potassium phosphate concentration of 2.5 g/l during the fermentation process of date wastes syrup did increase the citric acid production to 98.42 ± 1.41 g/l. For the production of α-amylase, the obtained result shows that the presence of starch strongly induces the production of α-amylase with a maximum at 5.0 g/l. Among the various nitrogen sources tested, urea at 5.0 g/l gave the maximum biomass and α-amylase estimated at 5.76 ± 0.56 g/l and 2,304.19 ± 31.08 μmol/l/min, respectively after 72 h incubation at 30°C, with an initial pH of 6.0 and potassium phosphate concentration of 6.0 g/l.     

Peroxidase,acid phosphatase,RNase and DNase activity and isoform patterns during <Emphasis Type="Italic">in vitro</Emphasis> rooting of <Emphasis Type="Italic">Petunia</Emphasis> × <Emphasis Type="Italic">hybrida</Emphasis> microshoots

Specific activities and isoform patterns of peroxidases, acid phosphatases, DNases and RNases were studied in relation to in vitro rooting of Petunia × hybrida microshoots in the presence of 4 μM indole-3-butyric acid (IBA). Specific activities of the above enzymes increased in the course of rooting. Rhizogenesis could be related with an increased specific activity of peroxidases during the initiation phase, in parallel with increased lignin content. Twelve peroxidases, six anionic (A1–A6) and six cationic (C1–C6), seven acid phosphatases (ACP1–ACP7), seven RNases (R1–R7) and four DNases (D1–D4) isoforms were detected following native PAGE. Variation in the number of the above isoforms and their quantity was observed during different stages of rooting. Particularly, A2, A3, C3, C4, C5, ACP2, R1, R2, R3, and D4 isoforms appeared after the induction phase and could be related to emergence of root primordia. Additionally, R3 and D4 could be associated with cell division and differentiation, since these are only expressed in rooted microshoots. Moreover, the higher number of roots in IBA-treated microshoots could be related to the higher expression of RNase and DNase isoforms during initiation and expression phases.