Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.     

Purification and characterization of an extracellular keratinase from a hornmeal-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MTCC (9102)

Native proteolytic microorganisms were isolated from the hornmeal, which is a product obtained by treatment of horns and hoofs with steam under high pressure. Keratinolytic activities of these organisms were screened in mineral salt medium with 1% hornmeal. Bacillus subtilis MTCC (9102), a keratinase-producing organism causing extensive degradation of hornmeal has been identified. Keratinase was purified (45-fold) by ion exchange, and gel filtration chromatography. Among the keratinases produced by the various organisms, keratinase from the Bacillus subtilis strain reported by us was found to have a molecular weight range between 64 and 69 kDa and high activity in the pH range between 5 and 7, with maximum activity at pH 6.0 and at an optimum temperature of 40°C. It remained stable up to 70°C. The keratinase activity was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and 1 10-phenanthroline, and remained unaffected by phenylmethanesulfonyl fluoride (PMSF, relative activity: 93%), whereas iodoacetamide inhibited considerably. Zinc, magnesium, calcium, manganese, and nickel were found to enhance the enzyme activity, whereas mercury and copper inhibited its activity completely. The keratinolytic metalloprotease from native Bacillus subtilis differed from the other serine proteases. It may have potential applications in the bioconversion of keratinous wastes and eco-friendly dehairing in the leather industry.     

Characterization of a thermostable alkaline phytase from Bacillus licheniformis ZJ-6 in Pichia pastoris

A novel neutral phytase gene (phyC) from Bacillus licheniformis was cloned and expressed in Pichia pastoris under the control of AOX1 promoter. The gene is 1,146 bp in size and encodes a polypeptide of 381 amino acids. The recombinant PhyCm (rePhyCm), driven by the Saccharomyces cerevisiae α-mating factor, was secreted into culture medium. After 0.5% methanol induction for 96 h, the activity of rePhyCm in culture supernatant reached 0.23 U/ml. The optimum temperature and pH of purified rePhyCm were 60°C and 7.5, respectively. The rePhyCm was stable in a wide pH range of 5.0–9.0, especially for alkaline pH. The residual activities of rePhyCm retained over 80% after being incubated at pH 5.0–9.0, 37°C for 1 h in the presence of 1 mM CaCl₂. Interestingly, supplemental Ca²⁺ upgraded both the thermostability and pH stability of rePhyCm. Substrate specificity of rePhyCm, effects of metal ions and chemicals on phytase activity were also investigated in current study.     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Purification and Characterization of a Bacterial Phytase Whose Properties Make it Exceptionally Useful as a Feed Supplement

A periplasmatic phytase from a bacterium isolated from Malaysian waste water was purified about 173-fold to apparent homogeneity with a recovery of 10% referred to the phytase activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 65°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K _M = 0.15 mmol/l and k _cat = 1164 s⁻¹ at pH 4.5 and 37°C. The purified enzyme was shown to be highly specific. Among the phosphorylated compounds tested, phytate was the only one which was significantly hydrolysed. Some properties such as considerable activity below pH 3.0, thermal stability and resistance to pepsin make the enzyme attractive for an application as a feed supplement.     

Purification and kinetics of a protease-resistant,neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition

Abstract

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca⁺², K⁺, and Co⁺² and it was inhibited by SDS and Mg⁺², Al⁺², and Fe⁺². Purified enzyme showed specificity to different salts of phytic acid and values of K_m and V_max were 0.293?mM and 11.49 nmoles s^?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

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.     

Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

Strain improvement and up scaling of phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 under submerged fermentation conditions

Combination of physical and chemical mutagenesis was used to isolate hyper secretory strains of Aspergillus niger NCIM 563 for phytase production. Phytase activity of mutant N-1 and N-79 was about 17 and 47% higher than the parent strain. In shake flask the productivity of phytase in parent, mutant N-1 and N-79 was 6,181, 7,619 and 9,523 IU/L per day, respectively. Up scaling of the fermentation from shake flask to 3 and 14 L New Brunswick fermenter was studied. After optimizing various fermentation parameters like aeration, agitation and carbon source in fermentation medium the fermentation time to achieve highest phytase activity was reduced considerably from 14 days in shake flask to 8 days in 14 L fermenter. Highest phytase activity of 80 IU/ml was obtained in 1% rice bran–3.5% glucose containing medium with aeration 0.2 vvm and agitation 550 rpm at room temperature on 8th day of fermentation. Addition of either bavistin (0.1%), penicillin (0.1%), formalin (0.2%) and sodium chloride (10%) in fermented broth were effective in retaining 100% phytase activity for 8 days at room temperature while these reagents along with methanol (50%) and ethanol (50%) confer 100% stability of phytase activity at 4°C till 20 days. Among various carriers used for application of phytase in feed, wheat bran and rice bran were superior to silica and calcium carbonate. Thermo stabilization studies indicate 100% protection of phytase activity in presence of 12% skim milk at 70°C, which will be useful for its spray drying.     

Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.     

Purification and characterization of a new glucoamylopullulanase from thermotolerant alkaliphilic Bacillus subtilis DR8806 of a hot mineral spring

We have introduced a novel glucoamylopullulanase from thermostable alkaliphilic Bacillus subtilis DR8806 from a hot mineral spring in Iran. The enzyme was purified by ion-exchange chromatography following to ammonium sulphate precipitation. The molecular weight of the purified enzyme was estimated to be 65.5 kDa using denaturing acrylamide gel electrophoresis. The enzyme showed high activity over a wide pH range, from pH 5.0 to pH 11.0 with the optimum pH 9.5. Our results also indicated an optimum temperature of the enzyme activity at 70 °C. These features justify the characteristics of the alkaliphilic and thermostable bacterial proteins and enzymes. The enzyme did not require calcium and showed extreme stability with regard to surfactants, including SDS and Triton X-100, and oxidizing agents such as H₂O_2. These features of the enzyme suggest a promising potential for application in laundry industry. Furthermore, the enzyme was active on pulullan by 68% relative to normal activity on starch. Such characteristics have not already been reported for this type of enzyme, hence we propose that this is a new alkalophilic and thermostable enzyme.     

A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent

A serine alkaline protease from a newly isolated alkaliphilic Bacillus altitudinis GVC11 was purified and characterized. The enzyme was purified to homogeneity by acetone precipitation, DEAE-cellulose anion exchange chromatography with 7.03-fold increase in specific activity and 15.25% recovery. The molecular weight of alkaline protease was estimated to be 28 kDa by SDS PAGE and activity was further assessed by zymogram analysis. The enzyme was highly active over a wide range of pH 8.5 to 12.5 with an optimum pH of 9.5. The optimum temperature of purified enzyme was 45 °C and Ca²⁺ further increased the thermal stability of the enzyme. The enzyme activity was enhanced by Ca²⁺ and Mg²⁺ and inhibited by Hg²⁺. The present study is the first report to examine and describe production of highly alkaline protease from Bacillus altitudinis and also its remarkable dehairing ability of goat hide in 18 h without disturbing the collagen and hair integrity.     

Phytase produced on citric byproducts: purification and characterization

Citric pulp is an agro-industrial residue from the citrus processing industry with low inorganic phosphorus content applied in animal feed. A new bioprocess was developed to produce and purify a new phytase generated on citric pulp fermentation by Aspergillus niger FS3. The phytase was purified by cationic-exchange, anionic-exchange chromatography and chromatofocusing steps. From SDS–PAGE analysis, the molecular weight of the purified phytase was calculated to be 108 kDa. The phytase had an optimum pH of 5.0–5.5 and an optimum temperature of 60°C. The phytase displayed high affinity for phytate, and the K _m was 0.52 mM. The purified phytase was sufficiently able to withstand pelleting temperatures, retaining sufficiently high phytate-degrading activity.     

Purification and Characterization of Trypsin-like Enzymes from <Emphasis Type="Italic">Neomysis japonica</Emphasis> Using BApNA as Substrate

Using N-α-benzoyl-l-arginine p-nitroanilide (BApNA) as substrate, trypsin-like enzymes (TLEs) were purified from mysis (Neomysis japonica) following two chromatographic steps, Sephacryl S100 HR gel filtration and Benzamidine-Sepharose 4B affinity. They presented a high stability in the raw material, retaining over 45% of the initial activity after 30 days of storage at pH 8.0, 45 °C. The purified TLEs had relative molecular mass between 32 kDa and 33 kDa. With higher stability and greater activity, they had similar stability and activity profiles (pH 6.0–11.0, 15–65 °C) as bovine trypsin but had a different optimum temperature (35 °C for trypsin and 45 °C for TLEs). Similar to bovine trypsin, the purified TLEs could be activated by Ca²⁺ and Mg²⁺. And the purified TLEs also showed similar inhibitory profiles as bovine trypsin with the exception of chicken egg ovomucoid (CEOM), an effective inhibitor of bovine trypsin but less so for purified TLEs. Having TLEs with physiological efficiency 3.6 times that of bovine trypsin, the use of mysis as a source for commercial production of TLEs is discussed.     

Purification and Properties of a Protease Produced by Bacillus Subtilis CN2 Isolated from a Vietnamese Fish Sauce

Bacillus subtilis CN2 isolated from a Vietnamese fish sauce produced a large quantity of an alkaline protease, when grown on a soy peptone medium. The protease was purified to an electrophoretically homogeneous state and crystallized in its pure condensed solution. The molecular weight was determined to be 27,636 Da, and the N-terminal amino acid sequence was AQSVPYGISQIKAPAL. The optimum pH and temperature were pH 10.0 and 50 °C, respectively. The protease was active over a wide pH range of pH 7.0–11.0, and also active over a broad temperature range of 30–60 °C. The enzyme was potently inhibited by 1 mM phenylmethanesulphonyl fluoride, but resistant to 1 mM sodium dodecyl sulphate (SDS). This revised version was published online in July 2006 with corrections to the Cover Date.     

The endoglucanase from <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BEC-1 bears halo-tolerant,acidophilic and dithiothreitol-stimulated enzyme activity

A Bacillus subtilis strain BEC-1 demonstrating high carboxymethylcellulose-degrading activity was isolated from the forest soil sample. In order to characterize the biochemical specialty of its cellulase, the endoglucanase gene egl173 was cloned from this strain and was expressed in Escherichia coli. The gene encoded a protein of 499 amino acids with a molecular weight of 64 kDa. The purified Egl173 could hydrolyze both soluble and insoluble celluloses with distinct activities. This enzyme showed the highest enzyme activity at pH 4, maintained at least 85% activity in the pH range of 3–7, displayed maximum activity at 60°C and was highly stable between 30 and 60°C. It was found that this endoglucanase was increasedly active and retained its high stability after incubation with 5 M NaCl or 3 M KCl for 24 h. Furthermore, after incubation with 10 mM of dithiothreitol, the enzyme activity was significantly enhanced (125% of the control level). In the presence of diverse metal ions (except mercury and manganese cations), organic solvents, surfactants (except SDS) and chelating agent, this enzyme kept more than 85% active. This halo-tolerant, acidophilic and highly stable endoglucanase is prospectively to be exploited as the advanced enzymatic product for diverse industrial applications.