Physicochemical characterization of a thermostable glycoprotein bioemulsifier from Solibacillus silvestris AM1

A novel estuarine bacterial strain, Solibacillus silvestris AM1, was found to produce an extracellular, multimeric glycoprotein bioemulsifier, termed AM1, with a MW of 200 kDa and containing 30 kDa monomeric subunits. The bioemulsifier contained 3.6% of the minor carbohydrate components galactose and ribose/xylose. LC/MS-MS of the 30 kDa subunit revealed its homology with a flagellin-like protein arranged in the form of fibers, as shown by transmission electron micrographs. This is the first report of a flagellin-like protein that exhibits bioemulsifier activity being produced from a member of the Solibacillus genus. Bioemulsifier AM1 has a high emulsification index of 62.5% with 10^?2 critical micellar dilution. It was found to be thermostable and active in the pH 5–9 and 0–5 M NaCl ranges. Moreover, AM1 formed stable emulsions with a broad range of solvents, including aliphatics, aromatic hydrocarbons and oils, performing better than the well-known bioemulsifier emulsan. Emulsions formed with trichlorobenzene and paraffin oil have a pseudoplastic non-Newtonian rheological property, as observed by particle size and shear stress analysis. AM1, an eco-friendly bioemulsifier, formed stable emulsions in varied physical conditions, and these attributes may prove to be advantageous in cosmetic, pharmaceutical and environmental applications.     

Emulsifying properties of a marine bacterial exopolysaccharide

Exopolysaccharide produced by a marine Enterobacter cloaceae (designated as EPS 71a) emulsified hexane, benzene, xylene, kerosene, paraffin oil, cottonseed oil, coconut oil, jojoba oil, castor oil, groundnut oil and sunflower oil. However, it could form stable emulsions with groundnut oil and hexane at optimal concentration of 1 mg ml⁻¹. Further increase in concentration of EPS 71a did not show noteworthy increase in emulsification indices. Emulsions with groundnut oil and hexane were stable up to 10 days between pH 2 and 10 and in the presence of sodium chloride in the range of 5–50 mg ml⁻¹ at 35–37 °C. EPS 71a formed stable emulsion with xylene as compared to commercial gums such as arabic, tragacanth, karaya and xanthan.     

Characterization and flocculating properties of an extracellular biopolymer produced from a Bacillus subtilis DYU1 isolate

Biopolymer DYU500 produced from Bacillus subtilis DYU1 was found to have excellent flocculating ability. With the addition of 40 mg-DYU500/L and 50 mM CaCO₃, the optimum temperature for flocculation performance of DYU500 was 30 °C, giving the highest flocculating activity and rate of 13.5 and 97%, respectively. Analysis with Fourier transform infrared spectrophotometry (FT-IR), nuclear magnetic resonance spectrometry (NMR) and amino acid identification shows that the DYU500 biopolymer mainly possesses the structure of poly-glutamic acid (PGA). The average molecular weight of DYU500 was about (3.16–3.20) × 10⁶ Da as determined by gel permeation chromatography. The major components of biopolymer DYU500 were total sugars, uronic acids, proteins and polyamides (homopolymer of glutamic acid), accounting for a weight ratio of approximate 14.9, 2.7, 4.4 and 48.7% (w/w), respectively. The flocculating activity of DYU500 in the kaolin suspension was markedly stimulated by the addition of bivalent cations Ca²⁺ or Mg²⁺ in optimum concentration ranges of about 0.15–0.90 and 0.10–0.90 mM, respectively. The synergistic effect of cations was most effective at a weak acidic or neutral pH (6.0–7.0). The flocculating activity of DYU500 linearly decreased with an increase in incubation temperature and the activity was completely lost when heating upon 120 °C, arising from the destruction of the polyamides structure of DYU500. Moreover, mechanisms describing the flocculation process with DYU500 were proposed based on the experimental observations.     

Production and characterisation of exopolysaccharide from Streptomyces carpaticus isolated from marine sediments in Egypt and its effect on breast and colon cell lines

Twenty streptomycete strains were isolated from marine sediment samples collected from Nabq area, Sharm El-Sheikh, Red Sea Coast, Egypt. Four of them produce exopolysaccharides (EPS) showing marked in vitro antitumor activities. Morphological and cultural characteristics of the most significant strain (No. 3) were shown. Moreover, the sequence of this strain showed similarity with Streptomyces carpaticus. The results reveal that EPS produced by Streptomyces carpaticus No. 3 had high cytotoxicity reaching 51.7% and 59.1% against human tumor cells of breast and colon lines respectively. A chemical analysis of EPS indicated that the composing monosaccharides were galactouronic acid, glucose, xylose, galactose, mannose, and fructose with relative ratio of 3:1:1:2:2:1 respectively, with an average molecular weight (Mw) 1.180 × 10⁵?g/mol and of a number average molecular weight (Mn) 1.052 × 10⁵?g/mol. Also the EPS contained uronic acid (0.5072%) and monosaccharide sulphates (21.753%).     

Toxicity of basil and orange essential oils and their components against two coleopteran stored products insect pests

Two commercialized essential oils and their constituent compounds were investigated for fumigant and contact activities against two grain storage insects, adults of the maize weevil (Sitophilus zeamais) and the red flour beetle (Tribolium castaneum). The two commercialized basil and orange oils showed strong fumigant and contact activities against S. zeamais and T. castaneum. The constituents of the basil oil were linalool (21.83%), estragole (74.29%), and α-humulene (2.17%), and those of the orange oil were α-pinene (0.54%), sabinene (0.38%), β-myrcene (1.98%), limonene (96.5%), and linalool (0.6%). As a toxic fumigant, the basil oil was more effective (24-h LC₅₀ = 0.014 and 0.020 mg cm^− 3) than the orange oil (24-h LC₅₀ = 0.106 and 0.130 mg cm^− 3) against S. zeamais and T. castaneum adults, respectively. Among the constituents of the two essential oils, the toxicity of estragole was the highest (0.004 and 0.013), followed by linalool (0.016 and 0.023), limonene (0.122 and 0.171), α-pinene (0.264 and 0.273), and β-myrcene (0.274 and 0.275) based on 24-h LC₅₀ values (mg cm^− 3). Similar results were obtained in a contact toxicity test. The contact activity of basil oil was more toxic than orange oil, and estragole and linalool showed pronounced contact toxicity against S. zeamais and T. castaneum adults. Alpha-humulene had no activity as a fumigant at the tested doses, but it did have an effect as a contact poison, having 24-h LD₅₀ values of 0.040 and 0.045 mg adult^− 1 to S. zeamais and T. castaneum, respectively. Although basil oil, orange oil, and their components displayed both contact and fumigant toxicities, their effects were mainly exerted by fumigant action via the vapor phase. Thus, basil oil, orange oil, and their components could be potential candidates as new fumigants for the control of S. zeamais and T. castaneum adults.     

Synergistic effects of essential oil-based cream formulations against Culex quinquefasciatus Say and Aedes aegypti L. (Diptera: Culicidae)

Mosquitoes are major arthropod vectors responsible for several pathogenic diseases. In recent years, repellents of botanical origin, particularly essential oils, have been used against mosquitoes and have been found effective and safe. In this study, five different repellent cream formulations (CF1–5) were prepared using combinations of essential oils, including camphor, cinnamon, citronella, lemongrass, lime, orange, neem, basil, Vitex, Lantana, eucalyptus, and clove, and their repellency was tested using Culex quinquefasciatus Say and Aedes aegypti L. under laboratory conditions and compared to the standard synthetic repellent N,N-diethyl-meta-toluamide (DEET-12%, w/w). Among the five cream formulations, CF2 at a dose of 5 mg/cm² showed the longest protection time of 4.18 h and 3.31 h against C. quinquefasciatus and A. aegypti, respectively, under laboratory conditions. CF3 at a dose of 5 mg/cm² was moderately effective, with protection times of 3.42 h and 2.58 h against C. quinquefasciatus and A. aegypti, respectively, under laboratory conditions. CF2 at a dose of 5 mg/cm² was also tested in the field against wild mosquitoes for 3 h, and 100% protection was observed for the entire study period. Thus, CF2 could be used in developing an effective natural repellent as an alternative to the existing synthetic repellents to C. quinquefasciatus and A. aegypti.     

Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: Part I: Antioxidant capacity and antioxidant content

Response surface methodology was used to evaluate the quantitative effects of three independent variables: rapeseed moisture content, concentration of the added enzymes and conditioning temperature, on the antioxidant capacity and total phenolic, tocopherol, and phospholipid contents in the enzyme-treated rapeseed oils. The highest antioxidant capacity (1220.0, 964.8 μmol TE/100 g) total phenolic (83.3, 74.0 mg SA/100 g) and phospholipid (12,532, 12,376 mg/kg) contents reveal two rapeseed oils extruded from seeds contained 11% moisture, treated with cellulolytic and pectolytic enzymes (0.05%), respectively, and heated at 120 °C. However, the highest content of total tocopherols was determined in rapeseed oils pressed from seeds with 7% moisture, after addition of cellulolytic (0.05%) and pectolytic (0.1%) enzymes, heated at 90 and 105 °C, respectively. Total phenolic and phospholipid contents in the enzyme-treated rapeseed oils correlated significantly (p < 0.0000001) with antioxidant capacities of oils (R² = 0.8710 and 0.6581, respectively). Experimental results of the antioxidant capacity, total phenolic, tocopherol and phospholipid contents were close to the predicted values calculated from the polynomial response surface models equations (R² = 0.9727, 0.9870, 0.8390 and 0.9706 for the cellulolytic enzyme-assisted rapeseed oils and R² = 0.9148, 0.9489, 0.9426 and 0.9479 for the pectolytic enzyme-assisted rapeseed oils). The optimum rapeseed moisture content, enzyme concentration and conditioning temperature for the cellulolytic and pectolytic enzyme-treated rapeseed oils were 11% and 9.7%, 0.08% and 0.1%, and 120 °C, respectively.     

Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

Synthesis of thermo-sensitive copolymer with affinity butyl ligand and its application in lipase purification

In this study, thermo-sensitive N-alkyl substituted polyacrylamide polymer P_NNB was synthesized by using N-hydroxymethyl acrylamide(NHAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers, and its low critical solution temperature (LCST) was controlled to be 28 °C. The recovery of the thermo-sensitive polymer was over 98%. Butanol as a hydrophobic ligand was covalently attached onto polymer P_NNB and butyl ligand density was 80 μmol g^?1 polymer. The affinity polymer was used for purification of lipase from crude material. Optimized condition was pH 7.0, 35 °C adsorption temperature, 120 min adsorption time and 0.5 mg ml^?1 initial concentration of lipase. The adsorption isotherm accords with a typical Langmuir isotherm. The maximum adsorption capacity (Q_m) of the affinity polymer for lipase was 24.8 mg g^?1polymer. The affinity copolymer could be recycled by temperature-inducing precipitation and there was only about 6% loss of adsorption capacity after five recyclings. Specific activity of lipase was improved from 14 IU mg^?1 to 506 IU mg^?1 protein, and its recovery achieved 82%. The affinity polymer is suitable for the purification of target proteins from the crude material with large volume and dilute solution.     

Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri,and its biocontrol potential

Chitinase A (ChiA) produced by Bacillus thuringiensis subsp. colmeri 15A3 (Bt. 15A3) was expressed in Escherichia coli XL-Blue. The ChiA was purified using Sephadex G-200 and its molecular mass was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of chitinase activity on SDS-PAGE after protein renaturation indicated that the molecular mass of the protein band expressing chitinase activity was approximately 72 kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. ChiA has optimal activity at 50 °C and retains most of its activity between 20 and 60 °C. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The enzyme activity was significantly inhibited by Ag⁺ and Zn²⁺. ChiA significantly inhibited the spore germination of four species of fungi. The median inhibitory concentrations (IC₅₀) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian were 11.27 and 10.57 μg/ml, respectively. In surface contamination bioassays, the crude ChiA protein (12.6 mU) reduced the LC₅₀ (50% lethal concentration) of the crystal protein of Bt. 15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera.     

Response of native grasses and Cicer arietinum to soil polluted with mining wastes: Implications for the management of land adjacent to mine sites

Mine tailings are an environmental problem in Southern Spain because wind and water erosion of bare surfaces results in the dispersal of toxic metals over nearby urban or agricultural areas. Revegetation with tolerant native species may reduce this risk. We grew two grasses, Lygeum spartum and Piptatherum miliaceum, and the crop species Cicer arietinum (chickpea) under controlled conditions in pots containing a mine tailings mixed into non-polluted soil to give treatments of 0%, 25%, 50%, 75% and 100% mine tailings. We tested a neutral (pH 7.4) mine tailings which contained high concentrations of Cd, Cu, Pb and Zn. Water-extractable metal concentrations increased in proportion to the amount of tailings added. The biomass of the two grasses decreased in proportion to the rate of neutral mine-tailing addition, while the biomass of C. arietinum only decreased in relation to the control treatment. Neutron radiography revealed that root development of C. arietinum was perturbed in soil amended with the neutral tailings compared to those of the control treatment, despite a lack of toxicity symptoms in the shoots. In all treatments and for all metals, the plants accumulated higher concentrations in the roots than in shoots. The highest concentrations occurred in the roots of P. miliaceum (2500 mg kg^?1 Pb, 146 mg kg^?1 Cd, 185 mg kg^?1 Cu, 2700 mg kg^?1 Zn). C. arietinum seeds had normal concentrations of Zn (70–90 mg kg^?1) and Cu (6–9 mg kg^?1). However, the Cd concentration in this species was ～1 mg kg^?1 in the seeds and 14.5 mg kg^?1 in shoots. Consumption of these plant species by cattle and wild fauna may present a risk of toxic metals entering the food chain.     

Production of a collagenase from Candida albicans URM3622

Culture conditions (pH, time, temperature, inoculum size, orbital agitation speed and substrate concentration) for an extracellular collagenase produced by Candida albicans URM3622 were studied using three experimental designs (one 2⁶⁻² fractionary factorial and two 2³ full factorial). The analysis of the 2⁶⁻² fractionary design data indicated that agitation speed and substrate concentration had the most significant effect on collagenase production. Based on these results, two successive 2³ full factorial design experiments were run in which the effects of substrate concentration, orbital agitation speed and pH were further studied. These two sets of experiments showed that all variables chosen were significant for the enzyme production, with the maximum collagenolytic activity of 6.8 ± 0.4 U achieved at pH 7.0 with an orbital agitation speed of 160 rpm and 2% substrate concentration. Maximum collagenolytic activity was observed at pH 8.2 and 45 °C. The collagenase was stable within a pH range of 7.2–8.2 and over a temperature range of 28–45 °C. These results clearly indicate that C. albicans URM3622 is a potential resource for collagenase production and could be of interest for pharmaceutical, cosmetic and food industry.     

Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for β-glucosidase production. After fermentation both enzymes were purified. The β-glucosidases produced by the strains A. pullulans and T. aurantiacus were most active at pH 4.0–4.5 and 4.5, with apparent optimum temperatures at 80 and 75 °C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5–9.5 against 4.5–6.5) and more thermostable (98% after 1 h at 75 °C against 98% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t_(1/2) at 80 °C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. β-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol⁻¹ for T. aurantiacus and A. pullulans, respectively. The result showed that β-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus.     

Larvicidal activity of Amyris balsamifera,Daucus carota and Pogostemon cablin essential oils and their components against Culex pipiens pallens

Larvicidal activities of Amyris balsamifera, Daucus carota, and Pogostemon cablin essential oils were tested against Culex pipiens pallens. All three oils showed 100% larvicidal activity against C. pipiens pallens at 0.1 mg/mL. Among the tested oils, the larvicidal activity of D. carota oil was the strongest followed by P. cablin and A. balsamifera. Four active compounds such as β-eudesmol, elemol, patchoulol, and carotol were isolated from the three oils by open column chromatography. These compounds showed > 90% mortality against C. pipiens pallens at 0.1 mg/mL. In acute toxicity testing of the water flea, Daphnia magna, P. cablin oil was the most toxic followed by A. balsamifera, and D. carota. Among the isolated compounds, carotol was the most toxic to water fleas. The residues of P. cablin, A. balsamifera, and D. carota in water were 67.8%, 59.5%, and 51.2% at 2 days after treatment, respectively. High concentrations of elemol and patchoulol were detected 2 days after treatment compared to those of β-eudesmol and elemol. Whole oils and compounds tested were detected at < 50% after 7 days in water.     

Description of a new anaerobic thermophilic bacterium,Thermoanaerobacterium butyriciformans sp. nov.

Strain USBA-019^T, an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019^T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4 × 3.0–7.0 μm. Optimal growth occurs at 50–55 °C (35–65 °C). Optimum pH is 5.0–5.5 (4.0–8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019^T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019^T, Th. thermostercoris DSM22141^T and Th. aotearoense DMS10170^T found DNA–DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019^T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019^T (=CMPUJ U-019^T = DSM 101588^T).     

Ochrosin,a new biosurfactant produced by halophilic Ochrobactrum sp. strain BS-206 (MTCC 5720): Purification,characterization and its biological evaluation

A new bioactive molecule characterized as 4-dimethylaminobenzaldehyde (designated as Ochrosin) was isolated from a halophilic Ochrobactrum sp. strain BS-206 (MTCC 5720). Ochrosin exhibited good antimicrobial activity against both Gram-positive and Gram-negative bacteria and different Candida strains with minimum inhibitory concentration (MIC) values ranging from 4.68 to 150 μg mL⁻¹. It also exhibited anti-adhesive activity against various bacterial pathogens. Ochrosin caused an increase in the reactive oxygen species (ROS) levels to 32.4% and 28% and increased the NAD⁺/NADH ratio by 2.61 and 2.26 in S. aureus and E. coli, respectively. Ochrosin appears to interfere with ergosterol biosynthetic pathway in different Candida strains as it reduced the ergosterol content. Ochrosin showed insecticidal activity against three major stored-product grain pests like Tribolium castaneum, Callosobruchus chinensis and Sitophilus oryzae with LC₅₀ values of 7.3, 8.9 and 7.6 μg cc⁻¹, respectively. The anti-feedant index (ED₅₀) values observed were 1.07 and 1.35 μg cm⁻² of leaf area against Achaea janata and Spodoptera litura, respectively. This is the first report on Ochrosin, a multifunctional biosurfactant, produced by Ochrobactrum sp. BS-206 exhibiting promising antimicrobial, anti-adhesive, anti-feedant and insecticidal activities.     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795

A novel extracellular α-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible α-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg⁻¹. Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 210 kDa and 480 kDa from non-denaturing gradient PAGE. This α-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 α-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 °C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F78 in more fields. Kinetic studies revealed a K_m and V_max of 2.9 mmol l⁻¹ and 246.1 μmol (mg min)⁻¹, respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of α-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries.