Cloning of D-glucose dehydrogenase with a narrow substrate specificity fromBacillus thuringiensis M15

We have cloned an ORF ofBacillus thuringiensis M15, which encodes a protein sharing high similarity with D-glucose dehydrogenase. A high-expression plasmid (pBtGDH) for the ORF was constructed.Escherichia coli JM 109 transformed with pBtGDH exhibited D-glucose dehydrogenase activity, and the enzyme was purified by 3 chromatographic steps to homogeneity with 6.9 fold and a final yield of 13%. The purified enzyme has highly narrow substrate specificity for glucose and 2-deoxy-D-glucose and showed no activity with any other sugars we tested. The properties of the purified enzyme were similar to those of the D-glucose dehydrogenase (BtGDH) that is mainly produced inB. thuringiensis M15. These results show that the cloned gene encodes BtGDH, as we previously reported. This is the first report to determine the sequence of the enzyme with narrow substrate specificity. BtGDH shows 89% sequence similarity with D-glucose dehydrogenase fromBacillus megaterium IWG3 (GDH-IWG3), which has broad substrate specificity. A comparative analysis between BtGDH and GDH-IWG3 will reveal the differences between them and show the narrow specific activity of BtGDH.     

Catalytic properties of sepharose-bound l-Alanine dehydrogenase from Bacillus cereus

(1) l-Alanine dehydrogenase from Bacillus cereus was purified by a two-step chromatographic procedure involving Cibacron-Blue 3G-A Sepharose 4B-CL, and Sepharose 6B-CL, and immobilized on CNBr-activated Sepharose 4B. (2) Following immobilization via two of the six subunits, l-alanine dehydrogenase retained 66% of the specific activity of the soluble enzyme. The affinity of the immobilized enzyme for NH₄⁺, pyruvate and l-alanine, was not different to that of the soluble form. The K_m of the Sepharose-bound l-alanine dehydrogenase for pyridine coenzymes was 6–8-times higher than in the soluble case. (3) The stability of l-alanine dehydrogenase towards urea or thermal denaturation was increased by immobilization. (4) The incubation at 37°C for 24 h of the immobilized l-alanine dehydrogenase with 3 M NH₄Cl/NH₄OH buffer (pH 9) released 70% of the enzyme. The specific activity and the affinity of the ‘solubilized’ l-alanine dehydrogenase for the pyridine coenzymes was the same as that obtained with the original, soluble l-alanine dehydrogenase.     

Properties of levansucrase fromBacillus circulans

A polysaccharide-producing bacterium was isolated from cane sugar. It was identified asBacillus circulans and produced levansucrase at pH and temperature optima of 5–7 and 40°C respectively. The enzyme is extracellular and inducible with sucrose. It possesses initial hydrolytic and transferase activities that can be altered by modifying reaction conditions. Levansucrase was recovered from the fermentation broth by extraction with polyethylene glycol (1500 Da). Further purification resulted in an enzyme with a molecular mass of 52 kDa and a pI of 4.7. At high sucrose concentration (300 mM), the transferase activity but not the hydrolase activity were inhibited. Levan increased the transferase activity but had no effect on the hydrolytic activity. The levansucrase had high transferase activity with maltose, galactose and lactose and moderate activity towards sorbitol and glycerol.     

Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.     

Hypophosphite oxidase fromBacillus caldolyticus

Bacillus caldolyticus can utilize phosphorus either as phosphate, phosphite, or hypophosphite. When cultures are supplied with PO₂ as the sole source of phosphorus, the hypophosphite is oxidized to phosphate, which accumulates in the medium prior to the beginning of the log phase, and is then metabolised during growth. Resting cell suspensions also have the ability to oxidise PO₂ to PO₄. The reaction is specific for hypophosphite: PO₃ is not oxidised to PO₄, regardless of whether the cells are grown in PO₃- or PO₂-medium. The hypophosphite oxidase works optimally between pH 7.0 to 7.5, with a temperature optimum at 75°C; theK _m for NaH₂PO₂ is 320 μM. Sonication of cells, followed by high-speed centrifugation and ammonium sulfate fractionation of the cell-free extract showed that the PO₂ oxidation, which is accompanied by the formation of NADH, requires at least three components: An ammonium sulfate fraction of the cell-free extract, the residue fraction containing the respiratory chain, and NAD as cofactor. Most probably a second cofactor, so far not characterized, is required to accomplish full activity.     

Quaternary organization of subunits in the L-leucine dehydrogenase from Bacillus cereus

L-Leucine dehydrogenase from Bacillus cereus was examined in the electron microscope. The quaternary structure reveals a molecule that is built up from 8 subunits, identical in mass, arranged in 2 layers which are oriented mainly in a staggered form. In each layer subunits are positioned at the vertices of a square, leaving free a central protein-deficient region of 2.6 nm in diameter. The enzyme measures 11.1 nm in diagonal and 9.0 nm in edge length. Mean subunit diameter is 4.0 nm. The overall shape is a cube, slightly compressed, with 90% edge length in height.     

Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase

The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

蜡样芽孢杆菌亮氨酸脱氢酶基因在大肠杆菌中的表达及重组酶性质

本研究采用PCR技术从蜡样芽孢杆菌Bacillus cereus基因组DNA中克隆出亮氨酸脱氢酶基因,构建重组表达质粒p ET28α(+)-ldh,实现在大肠杆菌中的高效表达,并分析重组亮氨酸脱氢酶的酶学性质。结果表明,从Bacillus cereus成功克隆的亮氨酸脱氢酶编码基因约为1 000 bp,表达的重组亮氨酸脱氢酶相对分子质量约为40 k Da。酶学研究结果表明:该酶的最适反应温度为37℃,其热稳定性好,30℃的半衰期长达330 h;最适反应p H为9.5;在p H 7.0~8.0的缓冲液中保存24 h后仍保持原有酶活力的80%以上;金属离子Fe2+对该酶具有明显的促进作用,而EDTA强烈抑制亮氨酸脱氢酶的活性。动力学分析结果表明该酶对底物NADH催化的Km和Vmax分别为0.635 mmol/L和1.54μmol/(L·min)。亮氨酸脱氢酶基因在大肠杆菌中的成功表达为手性氨基酸的生物合成提供了可能。     

Lactate dehydrogenase A promotes communication between carbohydrate catabolism and virulence in Bacillus cereus

The diarrheal potential of a Bacillus cereus strain is essentially dictated by the amount of secreted nonhemolytic enterotoxin (Nhe). Expression of genes encoding Nhe is regulated by several factors, including the metabolic state of the cells. To identify metabolic sensors that could promote communication between central metabolism and nhe expression, we compared four strains of the B. cereus group in terms of metabolic and nhe expression capacities. We performed growth performance measurements, metabolite analysis, and mRNA measurements of strains F4430/73, F4810/72, F837/76, and PA cultured under anoxic and fully oxic conditions. The results showed that expression levels of nhe and ldhA, which encodes lactate dehydrogenase A (LdhA), were correlated in both aerobically and anaerobically grown cells. We examined the role of LdhA in the F4430/73 strain by constructing an ldhA mutant. The ldhA mutation was more deleterious to anaerobically grown cells than to aerobically grown cells, causing growth limitation and strong deregulation of key fermentative genes. More importantly, the ldhA mutation downregulated enterotoxin gene expression under both anaerobiosis and aerobiosis, with a more pronounced effect under anaerobiosis. Therefore, LdhA was found to exert a major control on both fermentative growth and enterotoxin expression, and it is concluded that there is a direct link between fermentative metabolism and virulence in B. cereus. The data presented also provide evidence that LdhA-dependent regulation of enterotoxin gene expression is oxygen independent. This study is the first report to describe a role of a fermentative enzyme in virulence in B. cereus.     

Cell-type specificity of l-leucyl l-leucine methyl ester

l-Leucyl l-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent which is converted to a membranolytic compound by dipeptidyl peptidase I and kills human leukocytes such as CD8+ T cells and monocytes but not B cells. The reagent has also been used in mice on the assumption that the cell-type specificity to murine leukocytes is the same as that to human leukocytes. During study on the effect of LeuLeuOMe on antigen-driven IL-2 production using murine splenocytes as antigen-presenting cells, however, we noticed that murine B cells were sensitive to LeuLeuOMe. We therefore examined the cell-type specificity using murine splenocytes and peritoneal macrophages. Flow cytometric analysis revealed that the most sensitive cells to LeuLeuOMe were CD8+ cells and that CD19+ cells (B cells) were as sensitive as CD3+ cells (T cells). Murine splenic B cells, which were either positively or negatively sorted with a cell sorter, were also sensitive to LeuLeuOMe, whereas human peripheral blood B cells, which were positively sorted, were not. Peritoneal macrophages were the most insensitive to LeuLeuOMe. Thus, this study demonstrated that the cell-type specificity to murine leukocytes is different from that to human leukocytes.     

A phosphate-stimulated NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of central carbon metabolism, was studied in a Bacillus cereus strain isolated from the phosphate layer from Morocco. Enzymatic assays with cell extracts demonstrated that when grown on Luria-Bertani (LB) medium, B. cereus contains a major NAD+-dependent GAPDH activity and only traces of NADP+-dependent activity, but in cells grown on Pi-supplemented LB medium a strong increase of the NADP+-dependent activity, that became predominant, occurs concurrently with a GAPDH protein increase. Our results show that B. cereus possesses two GAPDH activities, namely NAD+- and NADP+-dependent, catalyzed by two enzymes with distinct coenzyme specificity and different phosphate regulation patterns. The finding of a phosphate-stimulated NADP+-dependent GAPDH in B. cereus indicates that this bacterium can modulate its primary carbon metabolism according to phosphate availability.     

Some physical properties of coat material fromBacillus stearothermophilus spores

Coat material fromBacillus stearothermophilus spores has been examined for the following properties: X-ray diffraction pattern, infrared absorption spectrum, mechanical strength, and melting temperatures of the crystalline regions. The X-ray diffraction pattern of the coat material is different from that of both α-and β-keratin. The high melting temperature of the crystalline material indicates that its bonding is more stable than that of α- or β-keratin. The mechanical strength of the coat material ?10⁹N/m² is shown to be high enough to allow the coat to support the internal pressure in bacterial spores. This pressure has been postulated to produce a partial dehydration, which increases the ability of bacterial spores to withstand high temperatures in water.     

Systems of l-leucine transport into Saccharomyces cerevisiae protoplasts

l-[¹⁴C]Leucine transport into Saccharomyces cerevisiae protoplasts involves two systems (1 and 2) with different kinetic parameters. The K_T values for these systems are of the same order as those for intact yeast cells. These results suggest that the proteins related to the affinity constants are located in the cytoplasmic membrane.     

Thermostable,salt-tolerant amylase fromBacillus sp. 64

A thermostable, salt-tolerant amylase was produced byBacillus sp. 64, with maximum amylase production (8.0 U/ml culture filtrate) after 24-h growth. Partially purified amylase was stable at 60°C for 30 min and 80% of the original activity was retained when incubated in 5m NaCl over 24 h. Starch or dextrin was the best carbon source and peptone the best nitrogen source for the production of the enzyme. Amylase was secreted over a wide pH range (5 to 11) with the maximum activity between pH 7 and 8. Ca²⁺ and Mg²⁺ stimulated growth and enzyme production.NCL Communication No. 5209.     

Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported. In aqueous solution the purifiedp-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl, 4.1. The PNB carboxy-esterase catalyzed rapid ester hydrolysis for simple organic esters such as PNB-acetate, benzyl acetate and -naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [³H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [³H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.Abbreviations used -ME -mercaptoethanol - Cf cefaclor - Cf nucleus-PNB - (6R, 7R) 7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Cp cephalexin - Cp-PNB p-nitrobenzyl carboxy-ester of cephalexin - DEPC diethyl, pyrocarbonate - DFP diisopropyl fluorophosphate - DMSO dimethyl sulfoxide - DNP diethylp-nitrophenyl phosphate - EDTA ethylenediaminetetraacetic acid - EGTA ethylene, glycol-bis(aminoethyl ether) - N,N,NN tetracetic acid - Lc loracarbef - Lc-PNB p-nitrobenzyl carboxy-ester of loracarbef - Lc nucleus-PNB - (6R, 7S) 7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Lys C an endoproteinase specifically cleaving at C terminal lysine residues - MW_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PNB p-nitrobenzyl - PNBCE p-nitrobenzyl carboxy-esterase - SDS sodium dodecyl sulfate     

Morphology of a new bacteriophage isolated fromBacillus larvae

New bacteriophage (BL2) was isolated from plaques ofBacillus larvae cultures. Its morphology is distinct from the earlier bacteriophage BLA.     

Characteristics of a highly thermostable neutral protease produced fromBacillus stearothermophilus

A highly thermostable neutral protease was found in culture filtrates ofBacillus stearothermophilus. The optimum reaction pH and temperature of this protease were 6.0 and 60°C, respectively, and 90% activity remained even after heat treatment at 90°C for 30 min. The protease was markedly inactivated by diisopropyl fluorophosphate, but EDTA and iodoacetic acid hardly affected it. The neutral protease therefore could be defined as a highly thermostable, neutral(-serine) protease.