A novel method of producing the pharmaceutical intermediate (R)-2-chloromandelic acid by bioconversion

(R)-2-Chloromandelic acid (R­CM) is one of the chiral building blocks used in the pharmaceutical industry. As a result of screening for microorganisms that asymmetrically hydrolyze racemic 2­chloromandelic acid methyl ester (CMM), Exophiala dermatitidis NBRC6857 was found to produce R­CM at optical purity of 97% ee. The esterase that produces R­CM, EstE, was purified from E. dermatitidis NBRC6857, and the optimal temperature and pH of EstE were 30°C and 7.0, respectively. The estE gene that encodes EstE was isolated and overexpressed in Escherichia coli JM109. The activity of recombinant E. coli JM109 cells overexpressing estE was 553 times higher than that of E. dermatitidis NBRC6857. R­CM was produced at conversion rate of 49% and at optical purity of 97% ee from 10% CMM with 0.45 mg-dry-cell/L recombinant E. coli JM109 cells. Based on these findings, R­CM production by bioconversion of CMM may be of interest for future industrial applications.     

Biological characterization of two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains toxic against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT₅₀. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.     

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.Non-standard abbreviations BSA bovine serum albumin - CAT chloramphenicol acetyltransferase - Cm^R chloramphenicol resistant - PAB Penassay broth - SDS sodiumdodecylsulfate - Tc^R tetracycline resistant     

Production of pig liver esterase in batch fermentation of E. coli Origami

The establishment of a fermentation process for the production of pig liver esterase (PLE) in high yields is necessary for industrial applications. In our previous studies, we reported the recombinant expression of PLE in Escherichia coli Origami™ (DE3) in shake flask. Only a coexpression with chaperones GroEL/ES allowed the production of soluble and active enzyme. The optimization of the cultivation conditions, such as temperature, inducer concentrations, or media compositions to increase enzyme yield in a fermentation process is described here. Using fed-batch fermentation cell densities up to OD = 50 were obtained, but almost no active enzyme was expressed. Only batch fermentation was found suitable for production of active pig liver esterase and cell densities between OD = 7–13 and activities of 300–400 U L⁻¹ for isoenzyme PLE-1 (γPLE) and 1,400 U L⁻¹ for PLE-5 were obtained after 22 h total cultivation time or 18 h after induction of PLE expression, respectively.     

Effect of Feeding Bengal Gram (Cicer arietinum) at Different Protein Levels on Plasma Lipids of Rats

L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.     

Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.     

Identification of pig liver esterase variants by tandem mass spectroscopy analysis and their characterization

Pig liver esterase (PLE) is probably the most important carboxyl esterase in organic synthesis and is commercially obtained by extraction of the animal tissue. However, problems occur in its application due to the presence of several isoenzymes (α-, β- and γ-PLE). The functional expression of the γ-isoenzyme was already shown and differences in the enantioselectivity compared to the commercial preparations were confirmed. The amino acid and nucleotide sequences of the α- and β-PLE are still unknown. In this work, putative sequences of the α-isoenzyme were identified from a commercial PLE preparation by 2D gel electrophoresis, digestion with proteases and analysis using Matrix-assisted laser desorption/ionization–time of flight (TOF) and electrospray ionisation quadrupole–TOF mass spectrometry. Based on these results, three amino acid exchanges were introduced into the gene encoding γ-rPLE by site-directed mutagenesis, and the proteins were expressed in E. coli Origami (DE3). The produced PLE mutants were characterised with respect to their substrate specificity and enantioselectivity. No significant differences in the activity towards methyl butyrate were found, but several variants showed substantially enhanced enantioselectivity in the resolution of (R,S)-1-phenyl-2-butyl acetate with E = 100 for the best mutant V236P/A237G.     

Development of low-cost media for the culture of mosquito larvicides, Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis

Mosquito larvicides like Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis have been widely and effectively used in mosquito control programs, but the industrial production of these bacilli is expensive. Here we have attempted to develop three cost-effective media, based on cheap sources, potato, common sugar and bengalgram. Growth and production of the insecticidal proteins from these bacteria were satisfactory. Bioassay studies with different mosquito larvae showed considerable toxicity. Therefore the investigation suggests that potato-based culture media are more economical for the industrial production of B. sphaericus and B. thuringiensis serovar. israelensis.     

Characteristics and function of Alcaligenes sp. NBRC 14130 esterase catalysing the stereo‐selective hydrolysis of ethyl chrysanthemate

Aims: Alcaligenes sp. NBRC 14130 was found as a strain hydrolysing a mixture of (±)‐trans‐ and (±)‐cis ethyl chrysanthemates to (1R,3R)‐(+)‐trans‐chrysanthemic acid. The Alcaligenes cells also have hydrolytic activity for 6‐aminohexanoate‐cyclic dimer (6‐AHCD, 1,8‐diazacyclotetradecane‐2,9‐dione). The correlation of function on the enzyme from the Alcaligenes strain with hydrolysis activities for both ethyl chrysanthemate and 6‐AHCD was demonstrated. Methods and Results: The esterase was purified to homogeneity. The purified esterase hydrolysed 20 mmol l^?1 ester including the four stereoisomers to the corresponding (+)‐trans acid with a 37% molar conversion of ethyl (+)‐trans chrysanthemate. The esterase showed high hydrolytic activity for various short‐chain fatty acid esters, n‐hexane amide and 6‐AHCD. The amino acid sequence of the Alcaligenes esterase was identical to that of Arthrobacter 6‐AHCD hydrolase (EC 3.5.2.12) and similar to that of fatty acid amide hydrolase (EC 3.5.1.4) from Rattus norvegicus, having both serine and lysine residues of the catalytic site and the consensus motif Gly‐X‐Ser‐X‐Gly. Conclusion: The stereo‐selective hydrolytic activity was found in Alcaligenes sp. NBRC14130 by screening of ethyl chrysanthemate‐hydrolysing activity in micro‐organisms, and the purified esterase also acted on fatty acid esters and amides. Significance and Impact of the Study: This study has demonstrated that there are great differences in the enzymatic properties, amino acid sequence and catalytic motif of esterases in both Alcaligenes and Arthrobacter globiformis with excellent stereo‐selectivity for (+)‐trans‐ethyl chrysanthemate, but the amino acid sequence of Alcaligenes esterase is identical to that of Arthrobacter 6‐AHCD hydrolase.     

The 20-kDa chaperone-like protein of Bacillus thuringiensis ssp. israelensis enhances yield, crystal size and solubility of Cry3A

Aims: To determine whether the 20‐kDa chaperone‐like protein of Bacillus thuringiensis ssp. israelensis enhances synthesis, crystallization and solubility of the Cry3A coleopteran toxin and whether the crystalline inclusions produced are toxic to neonates of the Colorado potato beetle, Leptinotarsa decemlineata. Methods and Results: The cry3A gene was expressed in the 4Q7 strain of B. thuringiensis ssp. israelensis in the absence or presence of the 20‐kDa gene. The 20‐kDa protein enhanced Cry3A yield by 2·7‐fold per unit of fermentation medium. Crystal volumes averaged 2·123 and 0·964 μm³ when synthesized in, respectively, the presence or absence of the 20‐kDa protein. Both crystals were soluble at pH 5 and pH 6; however, the larger crystal was 1·7× and 1·5× more soluble at, respectively, pH 7 and pH 10. No significant difference in toxicity against L. decemlineata neonates was observed. Conclusions: This report demonstrated that the 20‐kDa chaperone‐like protein enhances yield, volume and solubility of the coleopteran Cry3A crystalline inclusions per unit crystal/spore mixture. Significance and Impact of the Study: This is the first report showing that an accessory protein (20‐kDa) could enhance synthesis and crystallization of Cry3A, a finding that could be beneficial for commercial production of this coleopteran‐specific insecticidal protein for microbial insecticides and possibly even for transgenic crops.     

Proteolysis, histopathological effects, and immunohistopathological localization of delta-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of lepidopteran olive tree pathogenic insect Prays oleae

Considering the fact that Prays oleae is one of the most pathogenic insects to the olive tree in the Mediterranean particularly in Tunisia, the mode of action of Cry insecticidal toxins of Bacillus thuringiensis kurstaki in Prays oleae midgut was investigated. The proteolysis of Bacillus thuringiensis δ-endotoxins in the midgut was a key step in determining their potency against Prays oleae. The latter's proteases activated the δ-endotoxins early, yielding stable toxins. The in vitro and in vivo binding of these toxins to Prays oleae larvae midgut was studied immunohistochemically, evidencing a midgut columnar cell vacuolization, microvilli damage, and then a pass of epithelium cell content into the larvae midgut. Moreover, Bacillus thuringiensis toxins were shown to bind to the apical microvilli of the midgut epithelial cells. The in vitro study of the interaction of Prays oleae midgut proteins with biotinylated Bacillus thuringiensis toxins allowed the prediction of four suitable receptor proteins in Prays oleae.     

Bacillus thuringiensis crystal protein (δ-endotoxin) gene expression is independent of early sporulation specific functions

Bacillus thuringiensis produces a parasporal insecticidal crystal protein. The correlation between sporulation and crystal protein production inBacillus thuringiensis var.israelensis was studied. The strain was made resistant’against streptomycin (St^R)-Acrystalliferous (Cry^-) cured derivatives and asporogenous acrystalliferous (Spo⁻ Cry⁻) mutants blocked at an early stage of sporulation were isolated. Plasmid transfer experiments were performed between St^R Spo⁺ Cry⁺ (streptomycin sensitive sporogeneous crystalliferous) and St^RR Spo⁺ Cry⁻ and also between St^s Spo⁺ Cry⁺ and St^R Spo⁻ Cry⁻ strains. StR colonies were selected. Insect toxicity was exhibited by the St^R isolates in both the cases. The process of crystal formation is, therefore, independent of early sporulative events.     

On the kinetics of the enzyme-catalyzed hydrolysis of axial chiral alkyl allenecarboxylates: preparation of optically active R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid and its optically pure S-(+)-methylester

Pig liver esterase (PLE) was used for the preparation of optically active alkyl allenecarboxylates with axial chirality. Free and immobilized enzymes were used as biocatalysts for the kinetic resolution of racemic ester substrates. Whereas the biotransformations using the free biocatalyst resulted in moderately to high enantiomeric ratios, the immobilization significantly decreased the E-value. The reaction conditions were optimized with respect to the enantiomeric ratio and scaled up. The enantiomeric ratio (E-value) was thereby enhanced by a factor of four to E=60. Under optimized conditions (free enzyme, addition of acetone as a cosolvent and Triton X-100 as an emulgator) in a preparative scale biotransformation, 282 mg of optically pure S-(+)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid methylester (96% ee, 82% yield) and 257 mg of R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid (83% ee, 80% yield) could be synthesized from the racemic substrate.     

Hydrogen and Polyhydroxybutyrate Producing Abilities of <Emphasis Type="Italic">Bacillus</Emphasis> spp. From Glucose in Two Stage System

Metabolic activities of four Bacillus strains to transform glucose into hydrogen (H₂) and polyhydroxybutyrate (PHB) in two stages were investigated in this study. Under batch culture conditions, Bacillus thuringiensis EGU45 and Bacillus cereus EGU44 evolved 1.67–1.92 mol H₂/mol glucose, respectively during the initial 3 days of incubation at 37°C. In the next 2 days, the residual glucose solutions along with B. thuringiensis EGU45 shaken at 200 rpm was found to produce PHB yield of 11.3% of dry cell mass. This is the first report among the non-photosynthetic microbes, where the Bacillus spp.—B. thuringiensis and B. cereus strains have been shown to produce H₂ and PHB in same medium under different conditions.     

Endospore dipicolinic acid detection during Bacillus thuringiensis culture

Aims: The aim of this work was to detect Bacillus thuringiensis endospore production during fermentation under conditions hindering endospore detection, i.e. in a complex undefined industrial medium with a high content of solids in suspension. Methods and Results: Bacterial endospore production was measured using the photoluminescence of dipicolinate (DPA) with Tb³⁺. The high temperature and pressure of a conventional autoclave was used to release DPA from the endospores. The endospore was obtained from B. thuringiensis var. kurstaki HD‐73 fermentations in industrial‐type media with 25·1 and 54·1 g l^?1 glucose, 4·4 and 35·3 g l^?1 soybean meal, 5·8 g l^?1 yeast extract, 9·2 g l^?1 corn steep solids and mineral salts. Conclusions: In this study, we successfully determined the DPA concentrations during the culture of B. thuringiensis in high‐concentration soybean meal media. A good correlation was found between microscope endospore counting and DPA measurements in the cultures. Significance and Impact of the Study: Because of synergy between Cry protein and endospore in B. thuringiensis bioinsecticides formulation, it is important to be able to determine endospore development during B. thuringiensis industrial‐type fermentation, in order to ascertain the beginning of sporulation.     

Construction of a bifunctional genetically labelled plasmid for Bacillus thuringiensis subsp. israelensis

A small cryptic plasmid of Bacillus thuringiensis subsp. israelensis was labelled in vitro with two genetic markers. One of the recombinant plasmids was mapped and transformed in Escherichia coli, Bacillus subtilis and Bacillus thuringiensis. This and similar shuttle plasmids could be very useful as vectors for the investigation of the toxin genes in their own host.Abbreviations BTI Bacillus thuringiensis subsp. israelensis - MDal megadaltons     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

Distribution Analysis of IS<Emphasis Type="Italic">231</Emphasis>-like Sequences Among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Serovars Inferred from Restriction Fragment Length Polymorphisms

The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-specific hybridization profiles. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding pattern similarities. Interestingly, intraserovar strains did not necessarily cluster together.     

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Summary A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.     

Enhancement of immune response by the proteinaceous crystal of Bacillus thuringiensis var thuringiensis

The proteinaceous crystal of $\text{Bacillus thuringiensis}$ var $\text{thuringiensis}$ was found to enhance humoral immune response in rats and guinea pigs immunised with sheep red blood cells. The enhancement was due to the increased levels of both 19S and 7S antibodies in the sera of the treated animals. A novel synthesis of 7S haemolytic antibodies was observed in case of crystal treated animals.