Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

A cost-effective medium for the large-scale production of Bacillus sphaericus H5a5b (VCRC B42) for mosquito control

Bacillus sphaericus has been widely used in mosquito control programs, but the large-scale production of this bacterium is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max) and peanut cake powder (Arachis hypogea) by using 100-l bioreactor. Sporulation, toxicity and biomass were satisfactory after B. sphaericus was produced on both media. Use of the soybean culture medium resulted in “maximum” toxicity (LC₅₀ 14.02 ng/ml against third instar Culex quinquefasciatus larvae), highest spore count (3.7 × 10⁹ spores/ml) and maximum biomass (4.6 g/l) within a short fermentation time of 21 h. Hence, this soybean-based culture medium was considered most economical for the large-scale industrial production of B. sphaericus.     

Growth of Thermus aquaticus and its TaqI endonuclease production

The culture behaviour of Thermus aquaticus was characterized. The response of the bacterium to various carbon (tryptone, glucose, glycerol) and nitrogen sources (yeast extract, NaNO₃, (NH₄)₂SO₄, leucine, thymine, thiamine, glutamic acid) was studied. Amino acids did not support growth, but CASTENHOLZ salt medium supplemented with yeast extract and glucose or tryptone resulted in good growth and production. A suitable medium composition giving the highest biomass concentration and enzyme yield was developed. The simple medium containing TYE-NaCl resulted in the highest biomass concentration, whereas CASTENHOLZ mineral medium supplemented with tryptone and yeast extract gave the highest specific activity and enzyme yield. The effect of inoculum age and size on growth was also investigated in order to improve the yield and process consistency. The use of shake flasks inoculated with precultures at their early or late stationary phase resulted in the same biomass concentration (0.56 ± 0.015 g/l) and similar maximum specific growth rates (0.258 ± 0.003 h^?1). Inoculum sizes between 1 and 2.5 per cent were optimal for cell growth. As the other papers on thermophilic microorganisms, including the T. aquaticus YT-1 strain, gave qualitative information on growth, the results presented here cannot be compared with others on a quantitative basis. TaqI endonuclease was purified using a 5 step protocol including cell disruption, adsorption, precipitation, column chromatography and final dialysis. The enriched fraction had a specific activity of 33,600 U TaqI endonuclease per mg protein.     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

Simple and efficient expression of <Emphasis Type="Italic">Agaricus meleagris</Emphasis> pyranose dehydrogenase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L⁻¹ PDH or 1,330 U L⁻¹ d⁻¹ in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.     

High Concentration—Ethanol Fermentation of Raw Ground Corn

1-Pyrroline-5-carboxylate dehydrogenase was purified and crystallized from Bacillus sphaericus. The crystalline preparation gave a single band on polyacrylamide slab gel electrophoresis. The molecular weight of the enzyme was determined to be about 100,000 by gel filtration. The enzyme consists of two subunits which are identical in molecular weight (50,000), as judged on SDS slab gel electrophoresis. The enzyme shows an optimum pH of 6.5 to 7.0. Its activity was 8.1 times higher with NADP⁺ than with NAD ⁺, and the enzyme was stabilized by NADP⁺. The apparent Km values for l-l-pyrroline-5-carboxylate, NADP⁺ and NAD⁺ are 4.2 × 10^–5m (with NADP⁺), 9.5 × 10~⁶m and 2.5 × IO^-3 m, respectively. The enzyme reaction is irreversible. A simple method for the determination of l-ornithine involving ornithine ¿-aminotransferase and 1- pyrroline-5-carboxylate dehydrogenase from B. sphaericus was developed. A linear relationship was found between the absorbance at 340 nm and the amount of l-ornithine (50 ~ 400 nmol), and between the fluorescence and the amount of l-ornithine (0.2 ~ 10 nmol).     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

Gene cloning,purification and characterization of thermostable alanine dehydrogenase of Bacillus stearothermophilus

The alanine dehydrogenase (l-alanine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.1) gene of Bacillus stearothermophilus IFO12550 was cloned and expressed in Escherichia coli C600 with a recombinant plasmid, pICD301, which was constructed from pBR322 and the alanine dehydrogenase gene derived from B. stearothermophilus. The enzyme overproduced in the clone was purified about 30 fold to homogeneity by heat treatment and two subsequent steps with a yield of 46%. The enzyme of E. coli-pICD301 was immunochemically identical with that of B. stearothermophilus. The enzyme has a molecular weight of about 240,000 and consists of six subunits identical in molecular weight (40,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 30 min; at 55°C and various pHs between 6.0 and 11.5 for 10 min. The enzymological properties are very similar to those of the mesophilic B. sphaericus enzyme (Ohshima, T. and Soda, K., Eur. J. Biochem., 100, 29–39, 1979) except for thermostability.     

Isolation and characterization of feather degrading enzymes from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site

The SN1 strain of Bacillus megaterium, isolated from soil of Ghazipur poultry waste site (India) produced extracellular caseinolytic and keratinolytic enzymes in basal media at 30°C, 160 rpm in the presence of 10% feather. Feathers were completely degraded after 72 h of incubation. The caesinolytic enzyme was separated from the basal media following ammonium sulphate precipitation and ion exchange chromatography. We report 29.3-fold purification of protease after Q Sepharose chromatography. The molecular weight of this enzyme was estimated to be 30 kDa as shown by SDS-PAGE and zymography studies. Protease activity increased by 2-fold in presence of 10 mM Mn²⁺ whereas Ba²⁺ and Hg²⁺ inhibited it. Ratio of milk clotting activity to caseinolytic activity was found to be 520.8 for the 30–60% ammonium sulphate fraction in presence of Mn²⁺ ion suggesting potential application in dairy industry. Keratinase was purified to 655.64 fold with specific activity of 544.7 U/mg protein and 12.4% recovery. We adopted the strategy of isolating the keratinolytic and caesinolytic producing microorganism by its selective growing in enriched media and found that feather protein can be metabolized for production of animal feed protein concentrates.     

Overproduction of alanine by Arthrobacter strains with glucose-nonrepressible L-alanine dehydrogenase

Summary Two Arthrobacter strains were identified as having high alanine productivity and L-alanine dehydrogenase (ADH) activity upon growth on glucose. They excreted large amounts of DL-alanine (37 and 81 g/1), but Bacillus sphaericus with glucose-repressible ADH did not at all. These results suggest that the glucose-nonrepressible ADH might be involved in alanine overproduction in the Arthrobacter strains.     

The induction of a dehydrogenase activity for branched chain amino acids in Bacillus subtilis

Summary In Bacillus subtilis a dehydrogenase activity for branched chain amino acids was induced twelvefold in glucose medium by isoleucine. To a lesser degree this activity was induced by metabolically related amino acids with the exception of leucine which hardly induced. The induced enzyme actvity is different from alanine dehydrogenase. The presumable role of this inducible enzyme in anteiso fatty acid biosynthesis is discussed.     

Evolution of resistance to the Bacillus sphaericus Bin toxin is phenotypically masked by combination with the mosquitocidal proteins of Bacillus thuringiensis subspecies israelensis

Two insecticidal bacteria are used as larvicides to control larvae of nuisance and vector mosquitoes in many countries, Bacillus thuringiensis ssp. israelensis and B. sphaericus. Field studies show both are effective, but serious resistance, as high as 50 000‐fold, has evolved where B. sphaericus is used against Culex mosquitoes. To improve efficacy and deal with even greater potential problems of resistance, we previously developed several recombinant larvicidal bacteria that combine the best mosquitocidal proteins of these bacteria. In the present study, we report laboratory selection studies using our best recombinant strain against larvae of Culex quinquefasciatus. This recombinant, Bti/BsBin, is a strain of B. thuringiensis ssp. israelensis engineered to produce a large amount of the B. sphaericus binary (Bin) toxin, which makes it more than 10‐fold as mosquitocidal as the its parental strains. Here we show that larvae exposed to Bti/BsBin failed to develop significant resistance after 30 successive generations of heavy selection pressure. The highest level of resistance obtained at the LC₉₅ level was 5.2‐fold, but declined to less than two‐fold at the 35th generation. Testing the selected populations against B. sphaericus alone showed resistance to Bin evolved, but was masked by combination with B. thuringiensis ssp. israelensis. These results suggest that recombinant bacterial strains have improved mosquito and vector management properties compared with the wild‐type strains used in current commercial formulations, and should prove useful in controlling important human diseases such as malaria and filariasis on a long‐term basis, even when used intensively under field conditions.     

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.     

Growth rate of a non-fermentative <Emphasis Type="Italic">Escherichia coli</Emphasis> strain is influenced by NAD<Superscript>+</Superscript> regeneration

By complementing a non-fermentative Escherichia coli (ldhA ⁻ pflB ⁻) strain with the recombinant Zymomonas mobilis ethanol pathway (pdc, adhB), we evaluated the effect of different levels of enzymatic activity on growth rate demonstrating that there is a direct relationship between anaerobic growth rate and the total specific activity of pyruvate decarboxylase, which is the limiting enzyme of this specific fermentative NAD⁺ regenerating pathway. This relationship was proved to be useful to establish a selection strategy based on growth rate for the analysis of lctE libraries, which encode lactate dehydrogenase from Bacillus subtilis.     

An improved method for the purification of Bacillus subtilis glucose dehydrogenase cloned in Escherichia coli

An improved and simplified purification procedure has been developed for the isolation of the Bacillus subtilis glucose dehydrogenase which has resulted in a 10 fold higher yield of pure enzyme. The purification procedure utilizes gene cloning and an additional ammonium sulfate step to facilitate the removal of contaminating proteins. The procedure requires fewer chromatographic steps than previously reported, thus simplifying the procedure. This improved and simplified purification of B. subtilis glucose dehydrogenase will facilitate further structure-function studies of this sporulation specific enzyme.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Occurrence of meso-alpha, epsilon-diaminopimelate dehydrogenase in Bacillus sphaericus.

A new amino acid dehydrogenase catalyzing the oxidative deamination of meso-α,?-diaminopimelate was found in the crude extract of Bacillus sphaericus IFO 3525. This dehydrogenase requiring NADP was specific for meso-diaminopimelate and the other isomers were not substrates. The enzyme was optimally active at about pH 10.5. NAD could not replace NADP.     

Economic production of Lysinibacillus sphaericus under solid state fermentation

A highly active mosquitocidal Lysinibacillus sphaericus namely Ls 9B24 was isolated from soil of Alexandria governorate in Egypt. It was more active than the standard strain, L. sphaericus 2362. The sporulation and toxin formation of both cultures grown on different leguminous seeds and by-products under solid state fermentation (SSF) were studied. Among the tested substrates, 6% cotton seed meal enhanced sporulation and the mosquitocidal activity of L. sphaericus 2362, while 6% fodder yeast enhanced sporulation and the mosquitocidal activity of Ls 9B24. The optimum SSF growth conditions for maximum mosquitocidal activity by both cultures were using coarse wheat bran as a carrier material, 50% initial moisture content, 4–64 × 10⁶ colony forming units (CFU)/g solid medium inoculum and 6 days’ incubation period at 30°C. Addition of 0.5% yeast extract enhanced toxicity about 2.2 and 1.8 fold for L. sphaericus 2362 and Ls 9B24, respectively.     

Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium

Summary Isolation and identification of a thermotolerant feather-degrading bacterial strain from Thai soil as well as purification and properties of its keratinase were investigated. The thermotolerant bacterium was identified as Bacillus licheniformis. The keratinase was purified to homogeneity by three-step chromatography. The purified enzyme exhibited a high specific activity (218 U mg⁻¹) with 86-fold purification and 25% yield. The enzyme was monomeric and had a molecular mass of 35 kDa. The optimum pH and temperature for the enzyme were 8.5 and 60 °C, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA and iodoacetamide, but was stimulated by metal ions. It hydrolysed soluble proteins with a relative activity of 4–100% and insoluble proteins, including keratins, with a relative activity of 3–35%. Therefore, the enzyme could improve the nutritional value of meat- and poultry-processing wastes containing keratins, collagen and gelatin.     

Aerobic production of alanine by<Emphasis Type="Italic"> Escherichia coli aceF ldhA</Emphasis> mutants expressing the<Emphasis Type="Italic"> Bacillus sphaericus alaD</Emphasis> gene

Alanine was produced from glucose in an Escherichia coli aceF ldhA double mutant strain that contained the pTrc99A-alaD plasmid expressing Bacillus sphaericus alanine dehydrogenase. The aceF gene encodes one of the proteins of the pyruvate dehydrogenase complex, and therefore this strain required acetate as an additional carbon source. The ldhA gene encodes fermentative lactate dehydrogenase, a competitor of alanine dehydrogenase for the substrate pyruvate. Fermentations included an oxygenated growth phase followed by an oxygen-limited alanine production phase. The lowest value for the mass transfer coefficient (k_La) studied during the production phase yielded the greatest alanine. With feeding of glucose and NH₄Cl, 32 g/l alanine accumulated in 27 h with a yield of 0.63 g alanine generated per gram glucose consumed.