Lime and Sulphide-Free Dehairing of Animal Skin Using Collagenase-Free Alkaline Protease from Vibrio metschnikovii NG155

The objective of this work was to isolate a microorganism producing alkaline protease that can be used as an ecofriendly alternative to chemicals in dehairing process of leather manufacture. Alkaline protease producing bacterium Vibrio metschnikovii NG155 was isolated from soil samples of leather industry. The protease was highly effective in dehairing of goat skin, completely eliminating the use of lime and sulfide. Histological studies of the skin after dehairing showed that the enzyme did not damage the collagen layer and brought good fiber opening. Absence of collagenase activity was confirmed by reacting pure collagen with the enzyme and analyzing it on SDS PAGE, which showed no degradation of collagen. The enzyme was stable in a wide range of pH (7–11) and temperature (10–50 °C), which makes it suitable for industrial application.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

Isolation and characterization of a virulent Vibrio sp. bacterium from clams (Meretrix meretrix) with mass mortality

MM5 was a bacterial strain isolated from moribund clam (Meretrix meretrix) collected from a farm with mass mortality outbreak. Primary genotypic and phenotypic identification including 16S rDNA sequence analysis, multilocus sequence analysis (MLSA) of four housekeeping genes (gapA, ftsZ, mreB and topA) and biochemical tests suggested that strain MM5 was a Vibrio species closest to but different from Vibrio furnissii. Our previous study indicated that MM5 could induce a high mortality of M. meretrix (Yue et al., 2010). Quantitative challenge test was performed in this study to further evaluate the pathogenic potential of MM5, which showed that at 84 h post-inoculation, the cumulative mortalities of the MM5-injected group were significantly higher than those of control groups (P < 0.05). Cytopathological and histopathological features of the clam infected by MM5 were carried out by transmission electron microscopy (TEM) and Hematoxylin and Eosin (H&E) staining, respectively. Cytopathologically, foci of MM5 were found in hepatocytes of the clam infected by MM5. In addition, cytopathological lesion was detected in foot of infected clam. Histopathologically, MM5 was detected in different tissues of infected clam, including hepatopancreas, mantle and gill. Challenge test combined with pathological features indicated that MM5 was virulent to M. meretrix.     

Description of Tylenchorhynchus qasimii sp. n. with a New Report of T. kegasawai from Pakistan

A new stunt nematode, from soil around the roots of coconut (Cocos nucifera L.) and rice (Oryza sativa L.) from Karachi, Pakistan, is described and illustrated as Tylenchorhynchus qasimii n. sp. This new species is characterized by having females with 3–4 head annules, anteriorly directed stylet knobs, absence of post anal extension, presence of rounded sperm filled spermatheca and conoid to bluntly rounded hemispherical tail terminus. Males are common. Also included is the record of T. kegasawai from around the roots of rice (O. sativa L.), a new report from Sindh, Pakistan.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Evaluation of in vitro free radical scavenging potential of Streptomyces sp. AM-S1 culture filtrate

The present study was carried out to determine the free radical scavenging potential of culture filtrate of Streptomyces sp. AM-S1. Antioxidant activity of culture filtrate, lyophilized culture filtrate and ethyl acetate extract of Streptomyces sp. AM-S1 was determined by various in vitro assays such as ferric reducing power assay, phosphomolybdenum reduction, DPPH and ABTS radical scavenging activities. The results revealed that the culture filtrate of Streptomyces sp. AM-S1 effectively scavenged DPPH (IC₅₀ 90.2 μl/ml) and ABTS (IC₅₀ 13.2 μl/ml) radicals in a concentration dependent manner. In all the assays, ethyl acetate extract registered higher antioxidant activity when compared with the lyophilized culture filtrate (LCF). In addition, ethyl acetate extract (1123.4 μmole Fe(II)/mg extract) exhibited higher ferric reducing activity than the standard BHA (814.4 μmole Fe(II)/mg extract). Further works are needed on the isolation and identification of antioxidant molecules from the ethyl acetate extract of Streptomyces sp. AM-S1 culture filtrate.     

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated <Emphasis Type="Italic">Bacillus</Emphasis> species Y

Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn²⁺, Co²⁺ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.     

The haemosporidian parasites of bats with description of Sprattiella alecto gen. nov., sp. nov

Four species of Haemoproteidae were found in Pteropus alecto Temminck, 1837 in Queensland, Australia: i) Johnsprentia copemani, Landau et al., 2012; ii) Sprattiella alecto gen. nov., sp. nov., characterised by schizonts in the renal vessels; iii) Hepatocystis levinei, Landau et al., 1985, originally described from Pteropus poliocephalus Temminck, 1825 and, experimentally from Culicoides nubeculosus and found in this new host and for which features of the hepatic schizonts are reported; iv) gametocytes of Hepatocystis sp. which are illustrated but cannot be assigned to a known species. A tentative interpretation of phylogenetic characters of haemosporidians of bats is provided from the morphology of the gametocytes and localisation of the tissue stages with respect to recent data on the phylogeny of bats.     

Application of protease from Nocardiopsis sp. as a laundry detergent additive

The application of protease as a laundry detergent additive from a newly isolated Nocardiopsis sp., isolated from a soil sample collected in Northeast Brazil is reported. The optimal pH and temperature for protease activity were pH 10.5 and 50 °C, respectively. The enzyme was stable in a long-term incubation, showed 73.5% of initial activity at pH 10.5 and 61.7% at pH 12.0 for 120 min. Approximately 60% of initial activity remained after 120 min at 50 °C or after 30 min at 80 °C. Almost 87% of enzyme activity was retained in the presence of 10% (v/v) of peroxide at 40 °C, after 1 h. The protease also was stable in the presence of oxidants and surfactants such as SDS, saponin, Tween 20 and Tween 80 after 30 min. In the presence of Omo^®, the enzyme retained 64% of its activity at 40 °C for 1 h. An increase in the proteolytic activity (6–17%) was observed with K⁺, Na⁺, and Mg⁺⁺ ions. At pH 8.0, the protease hydrolysed casein maximally (50 U/mg).     

Purification and characterization of fibrinolytic alkaline protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. BLB

Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH₄)₂SO₄ and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus.     

Ophthalmomyiasis caused by a Phormia sp. (Diptera: Calliphoridae) larva in an enucleated patient

Ophthalmomyiasis rarely occurs worldwide, and has not been reported in Korea. We present here a case of ophthalmomyiasis caused by Phormia sp. fly larva in an enucleated eye of a patient. In June 2010, a 50-year-old man was admitted to Dankook University Hospital for surgical excision of a malignant melanoma located in the right auricular area. He had a clinical history of enucleation of his right eye due to squamous cell carcinoma 5 years ago. During hospitalization, foreign body sensation developed in his right eye, and close examination revealed a fly larva inside the eye, which was evacuated. The larva was proved to be Phormia sp. based on the morphology of the posterior spiracle. Subsequently, no larva was found, and the postoperative course was uneventful without any complaints of further myiasis. This is the first case of ophthalmomyiasis among the literature in Korea, and also the first myiasis case caused by Phormia sp. in Korea.     

Diplogasteroides asiaticus n. sp. is Associated with Monochamus alternatus in Japan

Diplogasteroides asiaticus n. sp. is described and illustrated, and its molecular profile and phylogenetic status within the family Diplogastridae are inferred. Morphologically, the new species is characterized by its stomatal structure, a tube-like stoma with three small, rod-like dorsal teeth and two subventral ridges; a spicule clearly ventrally bent at 1/3 from the anterior end; a gubernaculum with a rounded anterior end and sharply pointed distal end in lateral view; nine pairs of genital papillae with an arrangement of <v1, (v2, v3d), C, v4, ad, ph, (v5, v6, v7), pd>; a short tail spike in males; and a well-developed receptaculum seminis, i.e., the antiparallel blind sacs of the uteri beyond the vulva region and elongated conical tail in females. This new species is morphologically similar to D. haslacheri, but it can be distinguished by the morphology of the somewhat shorter tail in females. D. asiaticus n. sp. shares high sequence conservation with D. andrassyi as there is only one base pair difference in the nearly full-length 18S rDNA and seven base pair differences in the D2-D3 expansion segments of the 28S rDNA. Despite this sequence conservation, the species status of D. asiaticus n. sp. was confirmed using the biological species concept, as D. asiaticus n. sp. and D. andrassyi failed to generate viable F₂ progeny in hybridization tests.     

Descriptions of Eumonhystera borealis n. sp., Sphaerolaimus occidentalis n. sp., and a Redescription of S. gracilis de Man 1876 (Nemata) from Bothnian Bay,Baltic Sea

The free-living nematodes Eumonhystera borealis n. sp., Sphaerolaimus occidentalis n. sp., and S. gracilis de Man 1876 from Bothnian Bay in the northern Baltic Sea are described and illustrated. Eumonhystera borealis n. sp. differs from other species by its small body size (314-393 μm), narrow body (a = 37-49), and large anterior amphids. In Sphaerolaimus occidentalis n. sp. the amphids are posterior to the buccal cavity, and it differs from other similar species by having two sclerotized rings in the posterior part of the buccal cavity. An intersex is reported for S. gracilis. Sphaerolaimus gracilis is cannibalistic or a predator of other species, with a preference for E. borealis n. sp. Sphaerolaimus occidentalis n. sp. coexists with S. gracilis at depths of 80 m but not at 12 m.     

Description of Tunga bonneti n. sp. from Chile (Siphonaptera: Tungidae) with notes on specificity, chorology, dermecos and phenology

Smit (1968) misreported Tunga libis from Chile. Here we describe this flea as a new species. We analyzed free life females, male and embedded females. Moreover, we include data about its natural history and biology. These data will allow a better understanding about other Tunga fleas, and particularly for collecting males, which remain unknown to date.     

Description of Pratylenchoides camachoi n. sp. (Tylenchida: Pratylenchidae) from Spain

Pratylenchoides camachoi n. sp. from rosemary (Rosmarinus officinalis L.) in Sierra De Cazorla in southeastern Spain is described and illustrated. Pratylenchoides camachoi n. sp. is a bisexual species characterized by a cylindroid, sometimes clavate-shaped, male tail with a uniquely shaped smooth, rounded terminus. Females are characterized by a short esophageal overlap and six lines in the lateral field, which reduce to four in the tail region; not areolated except outer bands in the esophageal and tail regions.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

Prevalence of Haplorchis taichui and Haplorchoides sp. metacercariae in freshwater fish from water reservoirs, Chiang Mai, Thailand

A parasitological investigation on trematode metacercariae was made on 62 freshwater fishes of 13 species in northern Thailand; Cyclocheilichthys apogon, Puntioplites proctozysron, Labiobarbus siamensis, Barbodes gonionotus, Barbodes altus, Henicorhynchus siamensis, Osteochilus hasselti, Notopterus notopterus, Mystacoleucus marginatus, Anabas testudineus, Systomus orphoides, Morulius chrysophykadian, and Hampala macrolepidota. The fish were caught over the summer period (February-May 2007) from 2 Chiang Mai water reservoirs, i.e., the Mae Ngad (UTM 47Q E 503200, 47Q N 2119300) and the Mae Kuang Udomtara (UTM 47Q E 513000, 47Q N 2092600) Reservoirs in Chiang Mai province, Thailand. The prevalence of heterophyid (Haplorchis taichui and Haplorchoides sp.) metacercariae in these fish was 83.9% and 74.2% in the Mae Ngad and Mae Kuang Udomtara Reservoirs, respectively. The highest intensity of heterophyid metacercariae in H. siamensis in the Mae Ngad was 120.4 and that in P. proctozysron in the Mae Kuang Udomtara was 180.0. The fish, A. testudineus, C. apogon, and M. chrysophykadian, were not found to be infected with H. taichui metacercariae. The results show that the freshwater fish in Chiang Mai water reservoirs are heavily infected with H. taichui and Haplorchoides sp. metacercariae.     

Descriptions of Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina) from Haryana,India

Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and spermathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b’ = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c’ = 2.0 to 2.9, stylet = 11 to 12 µm, excretory pore in the region of median pharyngeal bulb, 43 to 47 µm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b’ = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c’ = 2.7 to 3.5, stylet = 6 to 7 µm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.     

Respiration Responses of a Caenorhabditis sp. and Aphelenchus avenae to the Nematicide, 1,2-dibromoethane (EDB)

The respiration rate of third stage larvae of Caenorhabditis sp. exposed to 0.53 × 10⁻² M EDB was 120% greater than in untreated checks and was highest shortly after the exposure began. Similarly-treated third- and fourth-stage Aphelenchus avenae exhibited no marked respiratory response. Different responses of these animals to EDB probably reflect basic physiological differences between the nematodes.     

Meloidogyne morocciensis n. sp. (Meloidogyninae), a Root-knot Nematode from Morocco

Meloidogyne morocciensis n. sp. is described from specimens parasitic on peach rootstock from Morocco. This species exhibits a combination of morphological characters similar to M. arenaria, M. incognita, and M. javanica. The perineal pattern of females is oval to squarish with a moderately high to high dorsal arch, and widely spaced, smooth striae; lateral lines are absent. The stylet, 16.5 μm long, has transversely ovoid, set-off knobs. Males have a set-off, annulated head region. The large, rounded labial disc is distinctly demarcated from the crescent-shaped medial lips; lateral lips are absent. The robust stylet, 24.6 μm long, has large, rounded knobs that taper slightly posteriorly. Mean second-stage juvenile (J2) length is 401 μm. The set-offhead region has incomplete annulations; the lip structures are dumbbell shaped. The stylet, 12.3 μm long, has rounded knobs that slope posteriorly. The J2 tail, 52.6 μm long, has irregularly sized annules in the posterior region and ends in a bluntly rounded tip. Tomato, tobacco, pepper, and watermelon are good hosts; cotton and peanut are not hosts. Meloidogyne morocciensis n. sp. reproduces by mitotic parthenogenesis and has a somatic chromosome number of 47-49. Its esterase phenotype is identical with the three-banded phenotype (A3) of M. arenaria.