Overexpression and characterization of a novel α-neoagarobiose hydrolase and its application in the production of D-galactonate from Gelidium amansii

An α-neoagarobiose hydrolase (α-NABH) from Cellulophaga sp. W5C, designated as AhgI, was identified, purified, and characterized. Its 1227 base pairs of coded sequence translate into a 408-amino acid protein that belongs to the GH117 family. Multiple sequence alignment of AhgI with other known α-NABHs showed 83% homology with AhgA from Zobellia galactanivorans. AhgI had an apparent molecular weight of 45 kDa and was highly active at pH 7.0 and 20 °C. The K_m and V_max values for neoagarobiose (NA2) were 1.03 mM and 10.22 U/mg, respectively. Apart from NA2, the enzyme showed activity against other neoagaro-oligosaccharides such as neoagarotetraose (NA4) and neoagarohexaose (NA6). AhgI was then employed in a prototype process to produce D-galactonate from Gelidium amansii. Agar from G. amansii was hydrothermally extracted and then enzymatically hydrolyzed by sequential addition of β-agarases and AhgI. The final hydrolysate containing D-galactose was then utilized for the microbial production of D-galactonate. This is believed to be the first report on the identification and characterization of an α-NABH derived from Cellulophaga species and its subsequent application in the synthesis of a value-added chemical directly from marine macroalgae.     

Differences in the photoacclimation and photoprotection exhibited by two species of the ciguatera causing dinoflagellate genus,Gambierdiscus

In culture, Gambierdiscus spp. have been shown to prefer irradiances that are relatively low (≤250 μmol photons m⁻² s⁻¹) versus those to which they are frequently exposed to in their natural environment (>500 μmol photons m⁻² s⁻¹). Although several behavioral strategies for coping with such irradiances have been suggested, it is unclear as to how these dinoflagellates do so on a physiological level. More specifically, how do long term exposures (30 days) affect cell size and cellular chlorophyll content, and what is the photosynthetic response to short term, high irradiance exposures (up to 1464 μmol photons m⁻² s⁻¹)? The results of this study reveal that cell size and chlorophyll content exhibited by G. carolinianus increased with acclimation to increasing photon flux density. Additionally, both G. carolinianus and G. silvae exhibited reduced photosynthetic efficiency when acclimated to increased photon flux density. Photosynthetic yield exhibited by G. silvae was greater than that for G. carolinianus across all acclimation irradiances. Although such differences were evident, both G. carolinianus and G. silvae appear to have adequate biochemical mechanisms to withstand exposure to irradiances exceeding 250 μmol photons m⁻² s⁻¹ for at least short periods of time following acclimation to irradiances of up to 150 μmol photons m⁻² s⁻¹.     

Influence of phosphorus nutrition on growth and metabolism of Duo grass (Duo festulolium)

Use of suitable plants that can extract and concentrate excess P from contaminated soil serves as an attractive method of phytoremediation. Plants vary in their potential to assimilate different organic and inorganic P-substrates. In this study, the response of Duo grass (Duo festulolium) to variable rates of soil-applied potassium dihydrogen phosphate (KH₂PO₄) on biomass yield and P uptake were studied. Duo grown for 5 weeks in soil with 2.5, 5 and 7.5 g KH₂PO₄ kg^?1 soil showed a significantly higher biomass and shoot P content of 8.3, 11.4 and 12.3 g P kg^?1 dry weight respectively compared to plants that received no soil added P. Also, the ability of Duo to metabolize different forms of P-substrates was determined by growing them in sterile Hoagland's agar media with different organic and inorganic P-substrates, viz. KH₂PO₄, glucose-1-phosphate (G1P), inositiol hexaphosphate (IHP), adenosine triphosphate (ATP) and adenosine monophosphate (AMP) for 2 weeks. Plants on agar media with different P-substrates also showed enhanced biomass yield and shoot P relative to no P control and the P uptake was in the order of ATP > KH₂PO₄ > G1P > IHP = AMP > no P control. The activities of both phytase (E.C.3.1.3.26) and acid phosphatases (E.C.3.1.3.2) were higher in all the P received plants than the control. Duo grass is capable of extracting P from the soil and also from the agar media and thus it can serve as possible candidate for phytoextraction of high P-soil.     

Cell-associated acid β-xylosidase production by Penicillium sclerotiorum

In recent decades, β-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 °C, under stationary condition for six days. The optimum activity temperature was 60 °C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 °C, with half-life of 240 and 232 min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The β-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V_max and K_m values was 0.48 μmol PNXP min^?1 mg^?1 protein and 0.75 mM, respectively. The enzyme was xylose tolerant with a K_i of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.     

Purification and characterization of two agarases from Agarivorans albus OAY02

Two extracellular β-agarases were purified from Agarivorans albus OAY2, which was isolated from seaweed collected in Qingdao, Shandong, China. The fermentation process was optimized with enzyme activity improving from 1.06 U/ml to 2.65 U/ml. Agarases were purified by ammonium sulfate fractionation and ion-exchange chromatography. Purification resulted in a 107-fold increase in agarase-a activity and a 52-fold increase in case of agarase-b, with high specific activities of 2715 and 1338 U/mg, respectively. The molecular masses of the agarases were estimated to be 50 kDa and 107 kDa, respectively. The optimum temperatures for the activities of agarase-a and agarase-b were 40 °C and 50 °C, respectively. Agarase-a was stable at 30 °C, while agarase-b was stable at 50 °C. The optimum pH values for agarase-a and agarase-b were both 9.0. Agarase-a was inhibited by Cu²⁺, Mn²⁺, Co²⁺ and EDTA, while agarase-b was inhibited by Cu²⁺, Mn²⁺ and Fe²⁺. The results of ¹³C NMR and TLC showed that hydrolysis of agarose by agarase-a produced neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6). Agarase-b hydrolyzed agarose to yield neoagarobiose (NA2) and neoagarotetraose (NA4). Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) and the enzymatic product results indicated that agarase-a and agarase-b might be novel agarases.     

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g⁻¹ dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g⁻¹ dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m⁻² s⁻¹ had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g⁻¹ dry weight in darkness to 20 mg g⁻¹ dry weight at a light intensity of 80 μmol photons m⁻² s⁻¹. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day⁻¹, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g⁻¹ dry weight was found at 65 μmol photons m⁻² s⁻¹. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.     

Oxidation of hydrogen sulphide in sour gas by Chlorobium limicola

The aim of this work was to assess the potential for bacterial oxidation of hydrogen sulphide as a purification method of sour gas. Using a continuous culture of Chlorobium limicola, high efficiencies of oxidation of both soluble and gaseous sulphide were achieved, with efficiencies for the latter exceeding 95%. Sulphide added as aqueous sodium sulphide was converted to sulphur and sulphate with almost total removal of the initial 100 mg S l⁻¹ within 24 h. Gaseous sulphide was oxidized at an efficiency of 95% (approximately 3 mmol S h⁻¹ (unit biomass Abs)⁻¹) over 1 h runs at a gas flow rate of 60 ml min⁻¹. With a sulphur recovery system to prevent sulphur accumulation, an efficiency of 70% was maintained. Biological removal of sulphide represents a potentially important biotechnological process, with high potential for viable scale up.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.     

Influence of whole microalgal cell immobilization and organic solvent on the bioconversion of androst-4-en-3,17-dione to testosterone by Nostoc muscorum

The use of free, immobilized and reused immobilized cells of the microalga Nostoc muscorum was studied for bioconversion of androst-4-en-3,17-dione (AD) to testosterone in hexadecane. Among polymers such as agar, agarose, κ-carrageenan, polyacrylamide, polyvinyl alcohol, and sodium alginate that were examined for cell entrapment, sodium alginate with a concentration of 2% (w/v) proved to be the proper matrix for N. muscorum cells immobilization. The bioconversion characteristics of immobilized whole algal cells at ranges of temperatures, substrate concentrations, and shaking speeds were studied followed by a comparison with those of free cells. The conditions were 30 °C, 0.5 g/L, and 100 rpm, respectively. The immobilized N. muscorum showed higher yield (72 ± 2.3%) than the free form (24 ± 1.3%) at the mentioned conditions. The bioconversion yield did not decrease during reuse of immobilized cells and remained high even after 5 batches of bioreactions while Na-alginate 3% was used; however, reuse of alginate 2% beads did not give a satisfactory result.     

Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes,antioxidants, and N-acetyl chitooligosaccharides

A chitinase (CHT), a chitosanase (CHS) and a protease (PRO) were purified from the culture supernatant of Serratia sp. TKU020 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHT, CHS and PRO determined by SDS-PAGE were approximately 65 kDa, 55 kDa and 55 kDa, respectively. CHT and CHS were inhibited by Mn²⁺, EDTA and PRO was inhibited by Mg²⁺, EDTA. The antioxidant activity of TKU020 culture supernatant was 78% (DPPH scavenging ability). N-Acetylglucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc)₂ were also produced from the culture supernatant by using TKU020 strain fermentation. The maximum production of GlcNAc and (GlcNAc)₂ was 1.3 mg/mL and 2.7 mg/mL, respectively, after 4 days of fermentation. With this method, we have shown that squid pen wastes can be utilized and it is effective in the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides, facilitating its potential use in industrial applications and functional foods.     

Optimization of the immobilization of sweet potato amylase using glutaraldehyde-agarose support. Characterization of the immobilized enzyme

A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1 U mg^?1 protein and 7.03 U mg^?1 protein for free and immobilized preparations, respectively).     

Growth of eight Gambierdiscus (Dinophyceae) species: Effects of temperature,salinity and irradiance

Little is known about how the growth of individual Gambierdiscus species responds to environmental factors. This study examined the effects of temperature (15–34 °C), salinity (15–41) and irradiance (2–664 μmol photons m⁻² s⁻¹) on growth of Gambierdiscus: G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus and G. ruetzleri and one putative new species, Gambierdiscus ribotype 2. Depending on species, temperatures where maximum growth occurred varied between 26.5 and 31.1 °C. The upper and lower thermal limits for all species were between 31–34 °C and 15–21 °C, respectively. The shapes of the temperature vs. growth curves indicated that even small differences of 1–2 °C notably affected growth potentials. Salinities where maximum growth occurred varied between 24.7 and 35, while the lowest salinities supporting growth ranged from <14 to 20.9. These data indicated that Gambierdiscus species are more tolerant of lower salinities than is generally appreciated. Growth of all species began to decline markedly as salinities exceed 35.1–39.4. The highest salinity tested in this study (41), however, was lethal to only one species, Gambierdiscus ribotype 2. The combined salinity data indicated that differences in salinity regimes may affect relative species abundances and distributions, particularly when salinities are <20 and >35. All eight Gambierdiscus species were adapted to relatively low light conditions, exhibiting growth maxima at 50–230 μmol photons m⁻² s⁻¹ and requiring only 6–17 μmol photons m⁻² s⁻¹ to maintain growth. These low light requirements indicate that Gambierdiscus growth can occur up to 150 m depth in tropical waters, with optimal light regimes often extending to 75 m. The combined temperature, salinity and light requirements of Gambierdiscus can be used to define latitudinal ranges and species-specific habitats, as well as to inform predictive models.     

Heterologous expression of a newly screened β-agarase from Alteromonas sp. GNUM1 in Escherichia coli and its application for agarose degradation

The gene of agaG1 from Alteromonas sp. GNUM1 encoding a β-agarase (AgaG1) was heterologously expressed in E. coli BL21 (DE3). The recombinant strain was cultured at 37 °C and then AgaG1 was expressed at 25 °C and 0.5 mM IPTG. The optimum conditions for AgaG1 to hydrolyze agarose were pH 7.0 and 40 °C. The main products of agarose hydrolysis by AgaG1 were confirmed to be neoagarobiose and neoagarotetraose. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively.     

Unmarked insertional inactivation in the gfo gene improves growth and ethanol production by Zymomonas mobilis ZM4 in sucrose without formation of sorbitol as a by-product,but yields opposite effects in high glucose

Sorbitol, one of the main by-products of growth on high sucrose concentrations, is catalyzed by glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28) in Zymomonas mobilis, which decreases the ethanol yield. In this study, an unmarked gfo mutant from Z. mobilis ZM4 was constructed using a site-specific FLP recombinase, and growth and ethanol production were evaluated with or without the addition of sorbitol to the media. The inactivation of gfo had contrasting effects in different substrates, especially at high concentrations. The maximum specific growth rate (μ_m) and theoretical ethanol yield value (Y_m) increased from 0.065 h⁻¹ and 60.56% to 0.094 h⁻¹ and 83.87% in 342 g/L sucrose, respectively. Conversely, in 200 g/L glucose, gfo inactivation decreased μ_m and Y_m from 0.15 h⁻¹ and 89.85% to 0.10 h⁻¹ and 67.59%, respectively, and prolonged the lag period from 16 h to 40 h. The addition of sorbitol slightly accelerated growth and sucrose hydrolysis by the gfo mutant in 342 g/L sucrose; however, addition of sorbitol restored the μ_m and Y_m of the gfo mutant in 200 g/L glucose to 0.14 h⁻¹ and 82.50%, respectively. Inactivation of gfo had a small effect on fructose utilization, and a positive one on mixture of glucose and fructose similar to that on sucrose. These results provide further understanding of the osmoregulation mechanisms in Z. mobilis and may help to exploit the biotechnological applications of this industrially important bacterium.     

Enzyme supplementation of wheat-based diets for broilers: 1. Effect on growth performance and intestinal viscosity

The influence of enzyme supplementation on performance and intestinal viscosity of male broiler chickens fed with diets containing high amount of wheat was examined in three experiments. In the first experiment, addition with an enzyme preparation including different cell wall degrading enzymes to diets containing 63 g kg⁻¹ and 72 g kg⁻¹ of wheat improved (P<0.05) feed conversion efficiency in the 72 g kg⁻¹ wheat diet. In addition, intestinal viscosity of chickens fed with the 72 g kg⁻¹ wheat diet was reduced (P<0.05). Weight gain and feed intake were not influenced by enzyme addition. In Experiments 2 and 3, the inclusion level of wheat in the diets was more than 80 g kg⁻¹ and four different enzyme preparations were used (two xylanase preparations, two mixed preparations). Overall, a significant effect on performance and intestinal viscosity of chickens was obtained as a result of enzyme supplementation in both experiments. In the first 21 days, improvements (P<0.05) in weight gain and feed conversion efficiency were found to be on average 5% and 6% in Experiment 2 and 7% and 8% in Experiment 3, respectively. When weight gain and feed conversion efficiency were examined on a weekly basis it was shown that the significant response of enzyme addition was confined to the first 4 weeks. However, the effect of enzyme supplementation was still significant in the whole period from 21–42 days. Feed intake was not influenced by enzyme addition. The viscosity of intestinal content in both the jejunum and ileum was in general reduced (P<0.05) with enzyme supplementation, the xylanase preparations proving to be the most efficient. It was concluded that enzyme supplementation of wheat-based diets resulted in improved performance of broiler chickens, which was related to a concomitant reduction in intestinal viscosity. However, the response of enzyme supplementation was most pronounced in diets with a wheat content higher than 80 g kg⁻¹.     

Purification and characterization of a periplasmic laccase produced by Sinorhizobium meliloti

Sinorhizobium meliloti CE52G strain produces a periplasmic laccase that has been purified by a two-step procedure involving heat treatment and immobilized metal affinity chromatography (IMAC). The fraction with laccase activity retained its original activity after 24 h of incubation at pH between 4.0 and 8.0 and after 3 h of incubation at 70 °C, pH 7.2 and supplemented with 1.3 M (NH₄)₂SO₄. It proved to be a homodimeric protein with an apparent molecular mass of 45 kDa each subunit and an isoelectric point of 6.2. CE52G laccase was inhibited by halides (NaF and NaCl), ions (Fe³⁺, Mn²⁺, and Cu²⁺), sulfhydryl organic compounds (β-mercaptoethanol and reduced glutathione), and electron flow inhibitors (NaCN and NaF). Laccase activity was strongly enhanced by (NH₄)₂SO₄, Na₂SO₄, and K₂SO₄. The effects of all these agents, as well as the probability of a partially unfolding polypeptide chain to enhance the interaction between the substrate and the active site, are discussed. CE52G laccase is a pH- and thermo-stable protein with promising biotechnological applications.     

Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins

Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photodynamic action but not considered the main cause of cell death. The in vivo effect of the photodynamic process on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein.Two cationic porphyrins (Tetra-Py⁺-Me and Tri-Py⁺-Me-PF) were used to photoinactivate E. coli (5.0 μM; 10⁸ cells mL^?1) and S. warneri (0.5 μM; 10⁸ cells mL^?1) upon white light irradiation at 4.0 mW cm^?2 for 270 min and 40 min, respectively. Total nucleic acids were extracted from photosensitized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double-stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry.E. coli was completely photoinactivated with both porphyrins (5.0 μM), whereas S. warneri was only completely inactivated by Tri-Py⁺-Me-PF (0.5 μM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA > 16S rRNA > genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5 min with Tri-Py⁺-Me-PF but almost unchanged with Tetra-Py⁺-Me after 40 min of irradiation. The amount of Tri-Py⁺-Me-PF bound to E. coli after washing the cells is higher than Tetra-Py⁺-Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells.     

α-Amylase from germinating soybean (Glycine max) seeds – Purification,characterization and sequential similarity of conserved and catalytic amino acid residues

Starch hydrolyzing amylase from germinated soybeans seeds (Glycine max) has been purified 400-fold to electrophoretic homogeneity with a final specific activity of 384 units/mg. SDS–PAGE of the final preparation revealed a single protein band of 100 kDa, whereas molecular mass was determined to be 84 kDa by MALDI–TOF and gel filtration on Superdex-200 (FPLC). The enzyme exhibited maximum activity at pH 5.5 and a pI value of 4.85. The energy of activation was determined to be 6.09 kcal/mol in the temperature range 25–85 °C. Apparent Michaelis constant (K_m(app)) for starch was 0.71 mg/mL and turnover number (k_cat) was 280 s^?1 in 50 mM sodium acetate buffer, pH 5.5. Thermal inactivation studies at 85 °C showed first-order kinetics with rate constant (k) equal to 0.0063 min^?1. Soybean α-amylase showed high specificity for its primary substrate starch. High similarity of soybean α-amylase with known amylases suggests that this α-amylase belongs to glycosyl hydrolase family 13. Cereal α-amylases have gained importance due to their compatibility for biotechnological applications. Wide availability and easy purification protocol make soybean as an attractive alternative for plant α-amylase. Soybean can be used as commercially viable source of α-amylase for various industrial applications.     

Bioconversion of post harvest crop residues and cattle shed manure into value-added products using earthworm Eudrilus eugeniae Kinberg

The post-harvest residues of some local crops, e.g. wheat (Triticum aestivum), millets (Penniseum typhoides and Sorghum vulgare), and a pulse (Vigna radiata) were subjected to recycle through vermicomposting by using the epigeic earthworm Eudrilus eugeniae Kinberg, under laboratory conditions. The crop residues were amended with animal dung; and three types of vermibeds were prepared: (i) millet straw (S. vulgare + Pennisenum typhoides in equal quantity) + sheep manure (1:2 ratio) (MS), (ii) pulse bran (Vigna radiata) + wheat straw (Triticum aestivum) + cow dung (1:1:2 ratio) (PWC), and (iii) mixed crop residues (mixing of all types crop residues, used in this study) + cow dung in 1:1 ratio (MCR + CD). The fourth treatment was cattle shed manure (CSM). At the end, ready vermicompost showed lower organic C content and higher concentrations of other important plant nutrients. Organic C content decreased in the order: MCR + CD (27.6%) > PWC (22.8%) > CMS (22.6%) > MS (19.4%). The ready vermicompost obtained from MCR + CD vermibed showed the maximum increase (% of initial level) in content of total N (143.4%), available P (111.1%) and exchangeable K (100.0%). The end product showed reduction in C:N ration between the ranges of 60.7% (CSM) and 70.3% (MCR + CD), at the end. The composting earthworm E. eugeniae exhibited the highest values of biological parameters: maximum mean individual biomass (1261.25 ± 7.0 mg), biomass gain (955.84 ± 11.03 mg), growth rate (10.62 ± 0.10 mg wt. worm⁻¹ day⁻¹), cocoon numbers (87.67 ± 6.51), and reproduction rate (0.66 ± 0.01 cocoons worm⁻¹ day⁻¹) in CSM container, while MS vermibeds showed the lowest values of these parameters. During experimentation, the maximum mortality for E. eugeniae was recorded in MS (16.67 ± 7.63%) followed by CSM > PWC > MCR + CD. Results indicated that the C:N ratio of the substrate drastically influenced the growth parameters of E. eugeniae, and it showed the close relations with maximum individual biomass gain (R² = 0.96), individual growth rate (R² = 0.82), and reproduction rate (cocoon worm⁻¹ day⁻¹) (R² = 0.72), in different treatments. This study clearly indicates that vermicomposting of crop residues and cattle shed wastes can not only produce a value-added product (vermicomposting) but at the same time acts as best culture medium for large-scale production of earthworms.     

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l^?1, pH 8) after 72 h at 30 °C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg^?1 and ～36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of Fe M^?1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20 g l^?1) to 3.9 μM ml^?1 of free amino groups in 24 h at an initial pH of 8.0, 30 °C and 120 rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.