Potential Anticoagulant Activity of Trypsin Inhibitor Purified from an Isolated Marine Bacterium <Emphasis Type="Italic">Oceanimonas</Emphasis> Sp. BPMS22 and its Kinetics

Protease inhibitors control major biological protease activities to maintain physiological homeostasis. Marine bacteria isolated from oligotrophic conditions could be taxonomically distinct, metabolically unique, and offers a wide variety of biochemicals. In the present investigation, marine sediments were screened for the potential bacteria that can produce trypsin inhibitors. A moderate halotolerant novel marine bacterial strain of Oceanimonas sp. BPMS22 was isolated, identified, and characterized. The effect of various process parameters like salt concentration, temperature, and pH was studied on the growth of the bacteria and production of trypsin inhibitor. Further, the trypsin inhibitor was purified to near homogeneity using anion exchange, size exclusion, and affinity chromatography. The purified trypsin inhibitor was found to competitively inhibit trypsin activity with an inhibition coefficient, K_i, of 3.44?±?0.13 μM and second-order association rate constant, k_ass, of 1.08?×?10³ M^?1 S^?1. The proteinaceous trypsin inhibitor had a molecular weight of approximately 30 kDa. The purified trypsin inhibitor showed anticoagulant activity on the human blood samples.     

Purification and Characterization of Microbial Protease Produced Extracellularly from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺ and K⁺, but 10 mM Fe³⁺ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).     

Serratia Protease

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease production of this strain was over 3 times as much as that of Serratia marcescens which was known as a protease-producing organism. The highly purified enzyme was prepared from the culture supernatant through ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75.

The purified enzyme moved homogeneously with a sedimentation constant, s_20,w of 3.8 S in ultracentrifugation and the molecular weight was determined to be 6.0 × 10₄ by the Archibald method. Determination of the ultraviolet absorption spectrum indicated the E^1%_{280 mμ,1 cm} was 13.0. Neither carbohydrate nor sulfur-containing amino acid was detected in the purified enzyme preparation. The enzyme showed maximal activity at pH 9.0 and at 40°C, and was stable under lower temperatures over the pH range from 5 to 10, whereas it was unstable at 37°C in alkaline conditions. The enzyme was completely inactivated by heating at 55°C for 15 min.     

Purification and Characterization of a Thermostable Neutral Metalloprotease I from Chloroflexus aurantiacus J-10-fl

Chloroflexus aurantiacus J-10-fl was found to contain two types (protease I and protease II) of thermostable proteases which were separated by Butyl-Toyopearl 650M chromatography. Protease I was purified to electrophoretic homogeneity from the culture broth of C. aurantiacus J-10-fl. The molecular mass of protease I was estimated to be approximately 66 kDa by SDS-PAGE, and the value of approximately 66kDa was also obtained by the Hedrick-Smith method, indicating that protease I was a monomer. The isoelectric point was 6.2. Protease I activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, and o-phenanthroline. The optimum pH for the activity of protease I was around 8.0. Addition of Ca²⁺ increased the pH and heat stabilities of protease I. The activity was stable between pH 4.0–11.0 and up to 75°C, and the maximum activity was observed at 70°C in the presence of 2mM CaCl₂. Protease I was resistant to the treatment by denaturing reagents (8 M urea or 1% SDS) at pH 8.0 and 20°C for 24 h. The sites of cleavage. in oxidized insulin B chain by protease I were similar to those by other microbial neutral metalloproteases. Elastase activity of protease I was not detected.     

Isolation and characterization of chromium(VI)-reducing bacteria from tannery effluents and solid wastes

In the present investigation, five novel Cr(VI) reducing bacteria were isolated from tannery effluents and solid wastes and identified as Kosakonia cowanii MKPF2, Klebsiella pneumonia MKPF5, Acinetobacter gerneri MKPF7, Klebsiella variicola MKPF8 and Serratia marcescens MKPF12 by 16S rDNA gene sequence analysis. The maximum tolerance concentration of Cr(VI) as K₂Cr₂O₇ of the bacterial isolates was varying up to 2000 mg/L. Among the investigated bacterial isolates, A. gerneri MKPF7 was best in terms of reduction rate. The optimum temperatures for growth and Cr(VI) reduction by the bacterial isolates were 35 and 40 °C, respectively except A. gerneri MKPF7 which grew and reduced Cr(VI) optimally at 40 °C. The optimum pH for growth and Cr(VI) reduction by K. cowanii MKPF2, A. gerneri MKPF7 and S. marcescens MKPF12 was 7.0 whereas the optimum pH for growth and Cr(VI) reduction by K. pneumoniae MKPF5 and K. variicola MKPF8 were 7.0, 8.0 and 6.0, 7.0, respectively. All the bacterial isolates showed maximum tolerance against Ni²⁺ and Zn²⁺ whereas minimum tolerance was observed against Hg²⁺ and Cd²⁺. The bacteria isolated in the present study thus can be used as eco-friendly biological expedients for the remediation and detoxification of Cr(VI) from the contaminated environments.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Degradation of chitin and production of bioactive materials by bioconversion of squid pens

Serratia marcescens TKU011, a protease- and chitosanase-producing bacterium, the optimized condition for protease and chitosanase production was found after the media were heated at 121 °C for 120 min and the culture was shaken at 25 °C for 5 days in 100 mL of medium containing 1% squid pen powder (SPP) (w/v), 0.1% K₂HPO₄, and 0.05% MgSO₄. An extracellular metalloprotease with novel properties of solvent stable, and alkaline was purified from the culture supernatant of S. marcescens TKU011 with squid pen wastes as the sole carbon/nitrogen source. The enzyme was a monomeric protease with a molecular mass of 48–50 kDa by SDS–PAGE and gel filtration chromatography. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU011 protease were 8, 50 °C, pH 5–11, and <40 °C, respectively. Besides protease and chitosanase, with this method, deproteinization of squid pen for β-chitin, the production of peptide and reducing sugar may be useful for biological applications.     

Comparative in situ remediation potential of Pseudomonas putida 710A and Commamonas aquatica 710B using plant (Vigna radiata (L.) wilczek) assay

A greenhouse study was conducted to determine the effect of cadmium (Cd)-resistant Pseudomonas putida strain 710A and Commamonas aquatica strain 710B, used either alone or as a binary mixture on the toxicity of cadmium to mungbean [Vigna radiata (L.) wilczek] plants. The bacterial strains 710A and 710B, isolated from the Semra mines in Ranchi, India, were resistant to 0.5 mM and 1 mM, CdCl₂ respectively. Moreover, both strains grew well at 10 °C and 30 °C. Of the two bacterial strains, strain 710B showed 129.6 and 83.5 times higher P solubilizing activity than strain 710A, when grown in liquid broth supplemented with cadmium at 10 °C and 30 °C, respectively. Furthermore, 16S ribosomal DNA (rDNA) sequencing identified 710A as a Pseudomonas putida strain and 710B as a Commamonas aquatica strain. The strain 710A significantly (p?<?0.05) stimulated the growth of roots (35.2 %) and shoots (30 %) of mungbean plants grown in soil treated with 110 μM CdCl₂. However, the increase in the case of 710B was found to be 15.4 % and 5.17 %, respectively. The loss of chlorophyll content was replenished by up to 80 %, 66 %, and 77.3 % by inoculation of 710A, 710B and binary mixture, respectively. Moreover the strains were able to reduce Cd accumulation in roots and shoots. Most significantly, residual Cd concentration in soil was reduced in the presence of bioinoculants. However, in terms of efficiency, the strains were found to be in the following order: 710A?>?710A +710B?>?710B. The results suggest that P. putida 710A strain is a potentially effective candidate metal to be used for sequestering and as a growth-promoting bioinoculant in Cd polluted soil.     

Appraising freeze-drying for storage of bacteria and their ready access in a rapid toxicity assessment assay

Direct toxicity assessment (DTA) techniques seek to measure the impact of toxic chemicals on biological materials resident in the environment. This study features the use of freeze-dried bacterial cells in combination with a rapid DTA analyser, SciTOX^?. The effects of three factors—cryoprotectant type, bacterial strain, and storage temperature—were tested in order to validate the shelf life of the freeze-dried cells. Three freeze-dried Gram-negative bacterial strains, Acinetobacter calcoaceticus, Escherichia coli and Pseudomonas putida, were tested by using the bacteria in the SciTox^? DTA assay and recording their responses to two standard toxicants: 2,4-dicholorophenol and 3,5-dichlorophenol. Each freeze-dried strain of bacteria was prepared in two forms—either pre-treatment with polyethylene glycol (PEG) or with sucrose/Tween 80—prior to storing at either 4 or ?20 °C for three different storage periods (1, 2 or 3 months). While the sucrose/Tween 80 pre-treated freeze-dried cells exhibited better cell viability, we concluded that PEG was a more suitable cryoprotectant for the bacteria used in the DTA assay because of EC₅₀ parity with fresh cell and zero-time freeze-dried cell assays. The results showed that freeze-dried cells, with appropriate materials and conditions, can give reproducible DTA results for up to 3 months. The availability of a biocomponent that can be activated by simple rehydration makes the deployment of this technology much easier for an end user.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Co-inoculation with <Emphasis Type="Italic">Enterobacter</Emphasis> and Rhizobacteria on Yield and Nutrient Uptake by Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) in the Alluvial Soil Under Indo-Gangetic Plain of India

The aim of this work was to evaluate the effects of co-inoculation with phosphate-solubilizing and nitrogen-fixing rhizobacteria on growth promotion, yield, and nutrient uptake by wheat. Out of twenty-five bacteria isolated from the rhizosphere soils of cereal, vegetable, and agro-forestry plants in eastern Uttar Pradesh, three superior most plant growth-promoting (PGP) isolates were characterized as Serratia marcescens, Microbacterium arborescens, and Enterobacter sp. based on their biochemical and 16S rDNA gene sequencing data and selected them for evaluating their PGP effects on growth and yield of wheat. Among them, Enterobacter sp. and M. arborescens fixed significantly higher amounts (9.32?±?0.57 and 8.89?±?0.58 mg Ng^?1 carbon oxidized, respectively) of atmospheric nitrogen and produced higher amounts (27.06?±?1.70 and 26.82?±?1.63 TP 100 µg mL^?1, respectively) of IAA in vitro compared to S. marcescens (8.32?±?0.39 mg Ng^?1 carbon oxidized and 21.29?±?0.99 TP 100 µg mL^?1). Although both M. arborescens and S. marcescens solubilized remarkable amounts of phosphate from tricalcium phosphate likely through production of organic acids, however, Enterobacter sp. was inactive. The effects of these three rhizobacteria were evaluated on wheat in alluvial soils of the Indo-Gangetic Plain by inoculation of plants with bacterial isolates either alone or in combinations in both pot and field conditions for two successive years. Rhizobacterial inoculation either alone or in consortium of varying combinations significantly (P?≤?0.05) increased growth and yield of wheat compared to mock inoculated controls. A consortium of two or three rhizobacterial isolates also significantly increased plant height, straw yield, grain yield, and test weight of wheat in both pot and field trials compared to single application of any of these isolates. Among the rhizobacterial treatment, co-inoculation of three rhizobacteria (Enterobacter, M. arborescens and S. marcescens) performed best in promotion of growth, yield, and nutrient (N, P, Cu, Zn, Mn, and Fe) uptake by wheat. Taken together, our results suggest that co-inoculation of Enterobacter with S. marcescens and M. arborescens could be used for preparation of an effective formulation of PGP consortium for eco-friendly and sustainable production of wheat.     

Downstream processing,characterization, and structure–function relationship of solvent‐, detergent‐, psychro‐, thermo‐, alkalistable metalloprotease from metal‐, solvent‐tolerant psychrotrophic Pseudomonas putida SKG‐1 isolate

The purification and characterization of psychro‐thermoalkalistable protease from psychrotrophic Pseudomonas putida isolate is being reported for the first time. A ～53 kDa protease was purified 21.4‐folds with 57.2% recovery by ultrafiltration and hydrophobic interaction chromatography. Kinetic analyses revealed the K_m and V_max to be 1.169 mg mL^?1 and 0.833 mg mL^?1 min^?1, respectively. The k_cat value of 3.05 × 10² s^?1 indicated high affinity and catalytic efficiency toward casein. The protease was most active at pH 9.5 and 40°C, with 100% stability in pH and temperature range of 6.0–11.0 and 10–40°C, respectively. Presence of Zn²⁺ increased the thermostability of protease (at 70°C) by 433%. Ethylene diamine tetra acetic acid (EDTA) and 1,10‐phenanthroline were inhibitory, whereas phenyl methyl sulfonyl fluoride (PMSF), p‐chloro mercuric benzoate (PCMB), and β‐mercaptoethanol were ineffective, revealing the enzyme to be a metalloprotease. Zinc, calcium, iron, nickel, and copper at 1 mM increased the enzyme activity (102–134%). Complete reversion of enzyme inhibition (caused by Ethylene diamine tetra acetic acid [EDTA]) by Zn²⁺ affirmed this enzyme as zinc‐dependent metalloprotease. At 0.1% concentration, Triton X‐100 and Tween 80 slightly increased, while SDS and H₂O₂ reduced the protease activity. In the presence of 0.1% commercial detergents, the enzyme was fairly stable (54–81%). In the presence of organic solvent, the protease was remarkably stable exhibiting 72–191% activities. In contrast, savinase exhibited good stability in the presence of hydrophilic solvents, while chymotrypsin showed elevated activities with benzene, toluene, and xylene only. Circular dichroism analysis revealed the protease as a β‐rich protein, having large fraction (～40%) of β‐sheets. Presence of different environmental conditions altered the β‐content, which accordingly affected the protease activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, <Emphasis Type="Italic">Bacillus iranensis (</Emphasis>X5B)

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48–50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50 % of activity at 2.5 M NaCl and about 70 % of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca²⁺. The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K _m and V _max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k _cat was obtained as 3.284 × 10^?2 s^?1. These special and important characteristics make this serine protease as valuable tool for industrial applications.     

Biochemical characterization of a halophilic,alkalithermophilic protease from <Emphasis Type="Italic">Alkalibacillus</Emphasis> sp. NM-Da2

An extracellular, halophilic, alkalithermophilic serine protease from the halo-alkaliphilic Alkalibacillus sp. NM-Da2 was purified to homogeneity by ethanol precipitation and anion-exchange chromatography. The purified protease was a monomeric enzyme with an approximate molecular mass of 35 kDa and exhibited maximal activity at 2.7 M NaCl, pH^55 °C 9 and 56 °C. The protease showed great temperature stability, retaining greater than 80 % of initial activity after 2 h incubation at 55 °C. The protease was also extremely pH tolerant, retaining 80 % of initial activity at pH^55 °C 10.5 after 30 min incubation. Protease hydrolyzed complex substrates, displaying activity on yeast extract, tryptone, casein, gelatin and peptone. Protease activity was inhibited at casein concentrations greater than 1.2 mg/mL. The enzyme was stable and active in 40 % (v/v) solutions of isopropanol, ethanol and benzene and was stable in the presence of the polysorbate surfactant Tween 80. Activity was stimulated with the oxidizing agent hydrogen peroxide. Inhibition with phenyl methylsulfonylfluoride indicates it is a serine protease. Synthetic saline wastewater treated with the protease showed 50 % protein removal after 5 h. Being halophilic, alkaliphilic and thermophilic, in addition to being resistant to organic solvents, this protease has potential for various applications in biotechnological and pharmaceutical industries.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Molecular Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in High-Temperature Petroleum Reservoirs

Anaerobic ammonium-oxidizing (anammox) process plays an important role in the nitrogen cycle of the worldwide anoxic and mesophilic habitats. Recently, the existence and activity of anammox bacteria have been detected in some thermophilic environments, but their existence in the geothermal subterranean oil reservoirs is still not reported. This study investigated the abundance, distribution and functional diversity of anammox bacteria in nine out of 17 high-temperature oil reservoirs by molecular ecology analysis. High concentration (5.31–39.2 mg l^?1) of ammonium was detected in the production water from these oilfields with temperatures between 55°C and 75°C. Both 16S rRNA and hzo molecular biomarkers indicated the occurrence of anammox bacteria in nine out of 17 samples. Most of 16S rRNA gene phylotypes are closely related to the known anammox bacterial genera Candidatus Brocadia, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia, while hzo gene phylotypes are closely related to the genera Candidatus Anammoxoglobus, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia. The total bacterial and anammox bacterial densities were 6.4?±?0.5?×?10³ to 2.0?±?0.18?×?10⁶ cells ml^?1 and 6.6?±?0.51?×?10² to 4.9?±?0.36?×?10⁴ cell ml^?1, respectively. The cluster I of 16S rRNA gene sequences showed distant identity (<92%) to the known Candidatus Scalindua species, inferring this cluster of anammox bacteria to be a new species, and a tentative name Candidatus “Scalindua sinooilfield” was proposed. The results extended the existence of anammox bacteria to the high-temperature oil reservoirs.     

Identification of a bacterium isolated from the diseased brown planthopper and determination of its insecticidal activity

The brown planthopper, Nilaparvata lugens, is one of the most harmful insect pests of rice crops in Asian countries. To find an effective biological control agent against this pest, we investigated the bacterial flora of field N. lugens collected from Jiangsu Province, China, in 2012 and tested its insecticidal activity. A novel bacterium strain, S-JS1, was isolated from N. lugens nymphs and adults and showed a high level of insecticidal activity. Based on its phenotypic, physiological and biochemical properties, and its 16S rRNA gene phylogeny, the isolate was assigned to Serratia marcescens; the name S. marcescens S-JS1 is proposed. The pathogenicity of S-JS1 against the third-instar nymphs, and the macropterous and the brachypterous adults of N. lugens were compared. The median lethal concentration (LC₅₀) values of S-JS1 against the brachypterous adult were the lowest (LC₅₀, 1.53?×?10⁸ colony forming units (cfu)/ml), followed against the macropterous adult (LC₅₀, 1.65?×?10⁹?cfu/ml) and third-instar nymphs (LC₅₀, 1.86?×?10⁹?cfu/ml) at 5 days post-infection. The median lethal time values of 8?×?10⁸?cfu/ml S. marcescens S-JS1 against the brachypterous adult, macropterous adult, and third-instar nymph were 4.5, 5.5, and 5.7 days, respectively. These results indicate that the S-JS1 isolate appears to be a promising S. marcescens strain with strong biocontrol potential against N. lugens.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

Optimization of protease production by Streptomyces sp. A6 using statistical approach for reclamation of shellfish waste

Protease producing Streptomyces sp. A6 was isolated from intertidal zone of the coast of Diu (Gujarat, India). Plackett–Burman method was applied to identify important factors (shrimp waste, FeCl₃, ZnSO₄ and pH) influencing protease production by Streptomyces sp. A6. Further optimization was done by response surface methodology using central composite design. The concentrations of medium components for higher protease production as optimized using the above approach were (g l^?1): Shrimp waste, 14; FeCl₃, 0.035; ZnSO₄, 0.065 and pH, 8.0. This statistical optimization approach led to production of 129.02 ± 2.03 U ml^?1 of protease which was 4.96 fold higher compared to that obtained using the unoptimized medium. The protease production was scaled to 3 l in a 5-l bench fermenter using optimized medium which further increased the production by 63.4%. Deproteinization and chitin recovery obtained at the end of fermentation was 85.12 ± 4.7 and 70.58 ± 1.33%, respectively. The present study is the first report on statistical optimization of medium components for production of protease by Streptomyces species using cheaper raw material such as shrimp waste. The study also explored the possibility Streptomyces sp. A6 for reclamation of shrimp wastes.     

Thermostable alkaline protease from Thermomyces lanuginosus: optimization,purification and characterization

A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-₁₀₀ and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K _m), maximal reaction velocity (V _max) and turnover number (K _cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s^?1, respectively. The best enzyme activators were K⁺, Ca²⁺ and Mn², respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry.