Isolation and characterization of thermostable collagen from the marine eel-fish (Evenchelys macrura)

Aim and methodsCollagen is the most abundant protein found in animal body, which is widely used for biomedical and pharmaceutical applications. In the present study, acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin wastes of marine eel fish (Evenchelys macrura) were isolated and characterized.ResultsASC and PSC extracted from eel fish skin showed the yields of 80 and 7.10 percent (based on dry weight), respectively. ASC and PSC comprising different α-chains (α1, α2 and α3) were characterized as type I and exhibited high solubility in acidic pH (1–4) and were soluble in the presence of NaCl at concentration up to 3.0 and 4.0 percent (w/v) for ASC and PSC, respectively. Amino acids analysis of both ASC and PSC contained imino acid of 190 and 200 residues per 1000 residues, respectively. The present results of ASC and PSC from eel fish skin exhibited higher thermal stability of 39 °C and 35 °C, respectively. Similar, Fourier transform infrared (FTIR) spectra of ASC and PSC were observed and suggesting that pepsin hydrolysis did not affect the secondary structure of collagen, especially triple-helical structure.ConclusionThese results suggest that the marine eel fish skin collagen close to the T_d (denaturation temperature) of mammalian collagen which could be used in the biomedical materials, food and pharmaceutical industries as an alternative source.     

Isolation and characterization of collagen from the cartilage of Amur sturgeon (Acipenser schrenckii)

The collagen in Amur sturgeon cartilage was isolated using sodium chloride (salt-solubilized collagen, SSC, 2.18%), followed by acetic acid (acid-solubilized collagen, ASC, 27.04%) and then pepsin (pepsin-solubilized collagen, PSC, 55.92%). These collagens appeared to be dense sheet-like film linked by random-coiled filaments under SEM. The denaturation and melting temperatures of PSC (35.71 and 123.90 °C) were significantly higher than SSC (32.64 and 114.51 °C) and ASC (32.98 and 120.72 °C) assessed by circular dichroism and differential scanning calorimetry, which could be attributed to its high imino acid content (22.57%) and degree of hydroxylation (47.29%). Electrophoresis pattern showed that the SSC and ASC were type I collagen, while PSC was predominantly type II collagen along with other minor types. Infrared spectra confirmed their triple helical structure, and indicated more hydrogen bonding in ASC and more intermolecular crosslinks in PSC. These results provide some basis for their large-scale production and further application as alternatives to mammalian collagen.     

Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar

Extraction of bromelain from pineapple peel (Nang Lae cultv.) using aqueous two phase system (ATPS) was optimized. Some biochemical properties including collagen hydrolysis were also investigated. Bromelain predominantly partitioned to the polyethylene glycol (PEG)-rich phase. The highest enzyme activity recovery (113.54%) and purification fold (2.23) were presented in the top phase of 15% PEG2000–14% MgSO₄. Protein pattern and activity staining showed the molecular weight (MW) of bromelain to be about 29 kDa. The extracted bromelain showed the highest relative activity at pH 7.0 and 55 °C. Its activity was decreased continuously by increasing NaCl concentration (up to 1.5% (w/v)). The bromelain extract was applied to hydrolyze the skin collagen of beef and giant catfish (0–0.3 units). The β, α₁, α₂ of giant catfish skin collagen extensively degraded into small peptides when treated with 0.02 units of the bromelain extract. Bovine collagen was hydrolyzed using higher bromelain up to 0.18 units. This study showed the ATPS can be employed to partially purify bromelain from Nang Lae pineapple peel and the enzyme effectively hydrolyzed the collagens.     

Influence of host plant nitrogen fertilization on hemolymph protein profiles of herbivore Spodoptera exigua and development of its endoparasitoid Cotesia marginiventris

Nitrogen has complex effects on plant–herbivore–parasitoid tritrophic interactions. The negative effects of low nitrogen fertilization in host plants on insect herbivores can be amplified to the higher trophic levels. In the present study, we examined the impact of varying nitrogen fertilization (42, 112, 196, and 280 ppm) of cotton plants (Gossypium hirsutum L.) on the interactions between the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), and the hymenopteran endoparasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae). We predicted that the development and fitness of C. marginiventris would be adversely affected by low host plant nitrogen fertilization through the herbivore S. exigua. The percentage of C. marginiventris offspring developing to emerge and spin a cocoon, and total mortality of parasitized S. exigua larvae were unaffected by nitrogen level. The developmental time of C. marginiventris larvae in S. exigua larvae feeding on low (42 ppm) nitrogen cotton plants was approximately 30% longer than that of those feeding on higher (112, 196, and 280 ppm) nitrogen plants. Parasitoid size (length of right metathoracic tibia), a proxy for fitness, of C. marginiventris males was positively affected by nitrogen level. Total amounts of S. exigua hemolymph proteins were not affected by nitrogen level, but were reduced by parasitism by C. marginiventris. Two proteins with molecular weights of ca. 84 and 170 kDa dominated the S. exigua larval hemolymph proteins. Concentrations of the 170 kDa hemolymph protein were unaffected by nitrogen treatment, but parasitism reduced concentrations of the 170 kDa protein. Concentrations of the 84 kDa protein, on the other hand, were interactively affected by parasitism and nitrogen treatment: higher nitrogen fertilization (112, 196, and 280 ppm) increased protein concentrations relative to the 42 ppm treatment for unparasitized S. exigua larvae, whereas nitrogen treatment had no effects on parasitized larvae. For S. exigua larvae feeding on 42 ppm nitrogen plants, parasitism increased concentration of the 84 kDa protein, while for those feeding on 112, 196, and 280 ppm nitrogen plants, parasitism decreased concentrations of the protein. Possible mechanisms and ecological consequences for the extended development of C. marginiventris on S. exigua hosts grown on low-nitrogen plants are discussed.     

Isolation,purification and characterization of pepsin soluble collagen isolated from silvertip shark (Carcharhinus albimarginatus) skeletal and head bone

Type II pepsin soluble collagens (PSC) were isolated from skeletal and head bone of silvertip shark; and examined for their biochemical and structural properties. Among the raw materials, the protein content (8.99%) was high in skeletal bone and the ash content (28%) was high in head bone. After the collagen extraction, the raw materials contained higher amount of ash content ranging from 82 to 88%. The hydroxyproline content of skeletal and skeletal PSC (30 and 113 mg/g) was higher than those head and head PSC. Both collagens were composed of two different α-chains (α₁- and α₂-chains) and were characterized as type II collagen. Amino acid analysis of skeletal and head PSC indicated imino acid contents of 156 and 175 amino acid residues per 1000 residues, respectively. Similar, Fourier transform infrared spectra of SCII and HCII were observed, which suggested that the isolation process did not affect the secondary structure and molecular order of collagen, particularly the triple–helical structure. Denaturation temperature of skeletal PSC (31 °C) was higher than that of head PSC. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. These results suggested that the PSC isolated from skeletal and head bone of silvertip shark were found to be suitable biomaterial in commercial applications as alternatives to mammalian collagen.     

Differences in the proportion of collagen and muscle in the canine lower urinary tract with regard to gonadal status and gender

Gonadectomy not only affects hormonal homeostasis but also alters the turnover of different components of the extracellular matrix in urogenital tissues. Collagen is an important component of the bladder and urethral walls and thus crucial for the mechanical properties of normal lower urinary tract (LUT) functions. This study aimed at investigating the possibility of differences in the proportion of collagen and muscle tissues in the LUT of intact and gonadectomised male and female dogs. Twenty clinically healthy dogs were used including 10 sexually intact dogs (5 males, 5 anoestrus females) and 10 gonadectomised dogs (4 males and 6 females). Four regions of the LUT, i.e. body and neck of the bladder as well as proximal and distal urethra were collected. The tissue sections were stained with Masson's Trichrome. Quantitative evaluation of the blue-stained area for collagen and red-counterstained area for muscle was performed using colour image analysis. The relative proportion of collagen and muscle significantly differed with the gonadal status, the gender and the region. Overall, gonadectomised dogs had a higher (P < 0.001) proportion of collagen and consequently a lower (P < 0.001) proportion of muscle than intact dogs. Regardless of gonadal statuses, females had a higher (P < 0.05) proportion of collagen and a lower (P < 0.05) proportion of muscle tissues than males. Gender differences were found in all four regions of the LUT in intact dogs but only in proximal urethra in gonadectomised dogs where spayed females had a higher (P < 0.05) proportion of collagen and less muscle (P < 0.05). Regional differences were observed in females; a higher proportion of collagen and therefore less muscle were found in the urethra compared with the bladder. Proportional differences in collagen and muscle between intact and gonadectomised animals suggest a relation of different hormonal statuses to structural changes in the canine LUT. Excessive collagen deposits and less muscular volume may impair structural and functional integrity of the LUT which may associate with the development of post-neutering urinary incontinence in the dog.     

Separation of lactobacilli bacteriocins from fermented broths using membranes

Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococci bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method.To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium (LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible microorganisms. Recovery yields using filtration were found to range between 53 and 68%, a high recovery performance.The bacteriocin activity of crude extracts of all the three lactobacilli were between 95 and 105 IU ml^?1. When the substances were separated using ultrafiltration membrane (4 kDa MWCO) their activity was enhanced to 145–150 IU ml^?1, achieving a total potency yield of 44–53%. Further enhancement of yields up to 36% was attained employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml^?1.Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields, optimising their activity as well as presenting a realistic option towards the formulation of these as commercially available antibacterial agents.     

Structural characterization of hemoglobins from Monilifera and Frenulata tubeworms (Siboglinids): First discovery of giant hexagonal-bilayer hemoglobin in the former “Pogonophora” group

Siboglinids are symbiotic polychete annelids having hemoglobins as essential oxygen- and sulfide-carriers for their endosymbiotic bacteria. We analyzed the structure of the hemoglobins from two species of siboglinids: the monilifera Sclerolinum contortum and the frenulata Oligobrachia webbi (i.e. haakonmosbiensis) from Norwegian cold seeps. Measured by Multi-Angle Laser Light Scattering (MALLS), Sclerolinum shows a 3190 ± 50 kDa hexagonal bilayer hemoglobin (HBL-Hb) and a 461 ± 46 kDa ring-Hb, just as vestimentifera, whereas Oligobrachia has a 409 ± 3.7 kDa ring-Hb only. Electrospray Ionization-Mass Spectrometry (ESI-MS) showed Sclerolinum HBL-Hb composed of seven monomeric globins (15–16 kDa), three disulfide-bonded globin heterodimers and three linkers. The heterodimers always contain globin-b (15814.4 ± 1.5 Da). Sclerolinum ring-Hb is composed of globins and dimers with identical masses as its HBL-Hb, but lacks linkers. Oligobrachia ring-Hb has three globin monomers (14–15 kDa) only, with no disulfide-bonded dimers. Comparison of Sclerolinum hemoglobins between Storegga and Haakon Mosby Mud Volcano, using the normalized height of deconvoluted ESI-MS peaks, shows differences in globin monomers abundances that could reflect genetic differences or differential gene expression between distinct seep populations. The discovery of HBL-Hb in Sclerolinum is a new element supporting the hypothesis of monilifera being phylogenetically more closely related to vestimentifera, than to frenulata.     

Characterization of a highly efficient heterodimeric xylosidase from Humicola insolens

A heterodimeric xylosidase (E.C. 3.2.1.37) with robust activity is secreted among the plant cell wall degrading enzymes produced by the saprophytic fungus Humicola insolens. The xylosidase has been purified to homogeneity by gel filtration and cation exchange chromatography, and demonstrated to be composed of two protein subunits of 68 and 17 kDa with a molecular mass in solution of approximately 85 kDa based on a combination of SDS-PAGE, size exclusion chromatography and analytical ultracentrifugation. Peptide sequence identities from the subunits indicate the 68 kDa subunit contains a catalytic protein domain and the 17 kDa subunit a carbohydrate binding module. The xylosidase has wide biotechnological potential with maximum activity exhibited at 70 °C and kinetic constants with p-nitrophenol xylopyranoside substrate that suggest it has the highest catalytic efficiency recorded to date (Vmax 22.17 μmoles/min/mg, Km 1.74 mM and Kcat 6787/s).     

Differential regulation of extracellular matrix constituents in myocardial remodeling with and without heart failure following pressure overload

Patients with aortic stenosis develop various degrees of myocardial hypertrophy and heart failure (HF) despite comparable transvalvular gradients. An important element in the transition from compensated hypertrophy to HF is dilatation of the left ventricle (LV). The molecular pathology associated with LV dilatation and development of HF is not known. Thus, we examined potential differences in the regulation of myocardial extracellular matrix (ECM) constituents in mice with hypertrophy only (ABnonHF) and with HF (ABHF) as response to comparable pressure overload. The ascending aorta was banded, or left loose in sham-operated mice. Increased lung weight and left atrial diameter indicating pulmonary congestion were used to identify ABHF mice. Cardiac function and geometry were evaluated by echocardiography. Despite comparable pressure gradients and cardiac output, ABHF had reduced fractional shortening (23%), reduced systolic (28%) and diastolic (32%) tissue velocity and increased LV internal dimension in diastole (10%) and systole (17%) (LVIDd/s) compared to ABnonHF (p ≤ 0.05). Microarray analyses identified 120 differently regulated genes related to ECM in ABHF compared to ABnonHF (p ≤ 0.05). Interestingly, in ABHF, we found a 24% (p ≤ 0.05) reduction of the LV collagen VIII protein levels despite increased levels of LV total collagen by 23% (p ≤ 0.05). LV collagen VIII correlated negatively with LVIDd (R = 0.55, p = 0.03) and LVIDs (R = 0.72, p = 0.002). As this protein may function as a “sealant” binding collagen fibrils together, reduction of collagen VIII could potentially contribute to LV dilatation and development of HF.     

Removal of residual methylene chloride from homoharringtonine by pre-treatment with ethanol

In this study, methylene chloride, which is a residual solvent in final purified homoharringtonine, was removed effectively through pre-treatment with ethanol. When the final HPLC-purified sample was concentrated using a rotary evaporator, the residual methanol easily met the ICH-specified value (3000 ppm), but methylene chloride did not meet the ICH-specified value (600 ppm). However, when the sample (methylene chloride: 10,000 ppm, methanol: 500 ppm) was concentrated through pre-treatment with 95% ethanol using a rotary evaporator, the residual methylene chloride easily met the ICH-specified value. Also, the residual ethanol (concentration > 10,000 ppm) was removed effectively below the ICH-specified value (5000 ppm) through microwave-assisted drying (microwave power: 400 W).     

Purification and characterization of a collagenolytic serine proteinase from the skeletal muscle of red sea bream (Pagrus major)

A collagenolytic serine proteinase (CSP) was purified from red sea bream (Pagrus major) skeletal muscle to homogeneity by ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Phenyl Sepharose and Hydroxyapatite. The molecular mass of CSP was approximately 85 kDa as estimated by SDS–PAGE and gel filtration. Optimum temperature and pH of CSP were 40 °C and 8.0, respectively. CSP was specifically inhibited by serine proteinase inhibitors, while inhibitors to other type proteinases did not show much inhibitory effects. The K_m and k_cat values of CSP for Boc-Leu-Lys-Arg-MCA were 3.58 µM and 0.13 s^? 1 at 37 °C, respectively. Furthermore, CSP hydrolyzed gelatin and native type I collagen effectively though its degradation on myosin heavy chain (MHC) was not significant, suggesting its involvement in the texture tenderization of fish muscle during the post-mortem stage.     

Toxicity of Acalypha indica (Euphorbiaceae) and Achyranthes aspera (Amaranthaceae) leaf extracts to Aedes aegypti (Diptera: Culicidae)

Alternative control strategies for the dengue vector Aedes aegypti L. (Diptera: Culicidae) include botanical insecticides. They are believed to pose little threat to the environment or to human health and may provide practical substitutes for synthetic insecticides. In this study, we determined the biological activities of methanol extracts of Acalypha indica L. (Euphorbiaceae) and Achyranthes aspera L (Amaranthaceae) leaves individually and in combination as botanical insecticides against Ae. aegypti. Based on LC₅₀ values for 4th instar Ae. aegypti, the combined extracts showed the strongest larvicidal activity (277 ppm). A. aspera and A. indica extracts individually gave similar results (409 and 420 ppm, respectively). Respective LC₅₀ values for pupae were 326 ppm, 456 ppm, and 467 ppm. In studies of smoke toxicity, 64% of females exposed to negative control smoke (no extract) blood fed on chicken, whereas 17% blood fed when exposed to smoke containing A. aspera extract and to positive control smoke (0.2% d-allethrin). In the field, treatment of water storage tanks (≈ 0.5 m³) with combined plant extract reduced larval and pupal populations by 97% and 81%, respectively, after 5 days. Given the results of this study, further evaluation of the combined (A. indica + A. aspera) extract as a mosquito larvicide is warranted. Mosquito coils with A. aspera extract also show promise as a practical and potentially economical means for mitigating mosquito blood feeding.     

Anthraquinone-based bird repellent for sunflower crops

Non-lethal alternatives are needed to manage bird damage to confectionery and oilseed sunflower crops (Helianthus annuus). Ring-necked pheasants (Phasianus colchicus) can cause localized damage to newly planted sunflower, and blackbirds (Icterids) damage ripening sunflower annually in the United States of America. We conducted seed germination experiments, a repellent efficacy study with ring-necked pheasants and Avipel^® repellent (a.i. 50% 9,10-anthraquinone), and laboratory and field efficacy studies with common grackles (Quiscalus quiscula) and Avipel^®-treated confectionery sunflower. Compared to the germination of seeds not treated with anthraquinone, we observed no negative effects of up to 12,223 ppm, 14,104 ppm, and 11,569 ppm anthraquinone seed treatments for germination of confectionery sunflower, oilseed sunflower, and canola seeds, respectively. Pheasants avoided emergent sunflower seedlings (12 days post-planting) from 15,800 ppm anthraquinone seed treatments during a caged preference test (P = 0.045). We observed a positive concentration–response relationship (P = 0.001) and predicted a threshold concentration (i.e., 80% repellency) of 9200 ppm anthraquinone for common grackles offered Avipel^®-treated confectionery sunflower seeds. Grackles also reliably discriminated between untreated sunflower and seeds treated with 1300 ppm anthraquinone in captivity (P < 0.001). During our field efficacy study for ripening confectionery sunflower, we observed 18% damage among anthraquinone-treated enclosures and 64% damage among untreated enclosures populated with common grackles (P < 0.001). Harvested seed mass averaged 2.54 kg (dry weight) among treated enclosures and 1.24 kg among untreated enclosures (P < 0.001). Our laboratory and field efficacy data provide a reliable basis for planning future field applications of anthraquinone-based repellents for protection of sunflower crops. Supplemental field efficacy studies are necessary for development of an effective avian repellent and management of avian depredation of ripening agricultural crops, including oilseed sunflower.     

Calcium protection of PS2 complex of Phaseolus coccineus from cadmium toxicity: In vitro study

The action of 10 and 20 mM Ca against harmful Cd effect on PS2 complex isolated from leaves of Phaseolus coccineus L. cv. Pi?kny Ja? was studied. The changes in fast chlorophyll a fluorescence induction kinetics and protein composition of PS2 complex were the symptoms of Cd toxicity and Ca protection of PS2 complex. Calcium applied at 10 mM concentration prevented F₀ reduction caused by the presence of 250–1000 μM Cd in the incubation mixture, but that of (the variable chlorophyll a fluorescence) F_v, F_m, F_v/F₀, and F_v/F_m only at 250 μM Cd. Ca concentration doubling in the incubation mixture resulted in complete overcoming the toxicity of 250–1000 μM Cd to F_v and F_m. However, the protection of F_v/F₀ and the photochemical efficiency of PS2 (F_v/F_m) from 1000 μM Cd was only partial even at 20 mM Ca. A protective effect of 10 mM Ca on D1, D2 and 17 kDa proteins was found in PS2 complex exposed to 250 μM Cd, and on 43 kDa protein in the complex treated with 500 μM Cd. However, 20 mM Ca counteracted the toxicity of 500 μM Cd to the 43, 47 and 17 kDa proteins, as well as the harmful effect of 1000 μM Cd on 47 and 17 kDa ones.     

Multi-chaperone-peptide-rich mixture from colo-carcinoma cells elicits potent anticancer immunity

Background: Chaperones play an important role in inducing anti-cancer immunity. To explore the probability of using chaperone-peptide-rich complexes extracted from colo-carcinoma cells as anti-cancer vaccine, we extracted and prepared chaperone-peptide-rich complexes from CT26 cells, which were subsequently investigated on anti-cancer efficacy. Methods: The crude extracts of the CT26 cells treated with heat and Trichosanthin were precipitated with salt and dialyzed to remove proteins below 50 kDa and above 300 kDa in molecular weight; the proteins with the molecular weights in 70 kDa, 90 kDa, 95 kDa, 110 kDa and 170 kDa were collected through gel filtration and SDS-PAGE. After confirmation, the purified proteins were used to determine their effects on lymphocyte proliferation, the activities of NK and CTL, tumor suppression and the tumor-bearing mouse survival. Results: The majority of the chaperone-peptides of anti-cancer immunity in CT26 cells, including HSP70-antigen peptide, HSP90-antigen peptide, gp96-antigen peptide, HSP-110 antigen peptide, HSP170-antigen peptide, was satisfactorily extracted that the multi-chaperone-peptide-rich mixtures were obtained. All the mixtures prepared could elicit lymphocyte proliferation, enhance the activities of CTL and NK, reinforce the tumor suppression and prolong the mouse survival. Conclusions: The multi-chaperone-peptide-rich mixtures could be prepared via dialysis and gel filtration combining with SDS-PAGE. Both the heat stress and Trichosanthin could induce and increase the mixtures, of which that treated by 42 °C heat and Trichosanthin was found to possess the strongest anti-cancer efficacy.     

Purification and characterization of aminopeptidase N from chicken intestine with potential application in debittering

An aminopeptidase with broad substrate specificity was purified to homogeneity (123.7-fold) with a yield of 3.43% from chicken (Gallus gallus) intestine using a combination of chromatographic separation strategies. The enzyme was identified as alanyl aminopeptidase or aminopeptidase N (APN) by Peptide Mass Fingerprinting. The molecular weight of the enzyme was estimated to be ∼180 kDa by SDS-PAGE and gel filtration chromatography. The enzyme was found to be a glycoprotein, having 40% sugar residue and a molecular mass of 108 kDa after deglycosylation. The enzymatic activity was optimal at 60 °C and pH 6.0. The enzyme preferentially hydrolyzed Leu-β-NA (K_m = 0.1 mM) followed by Ala, Phe, Tyr and Gly at N-terminal. The enzyme activity was completely inhibited by 1,10 phenanthroline (1 mM) and bestatin (1 mM) confirming it as a metalloprotease. Potential of this enzyme in combination with other endoproteases for the production of debittered protein hydrolysates has been discussed.     

In vitro characterization of CYP102G4 from Streptomyces cattleya: A self-sufficient P450 naturally producing indigo

Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV–Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily. It showed hydroxylation activity toward fatty acids generating ω-1, ω-2, and ω-3-hydroxyfatty acids, which is similar to the general substrate specificity of CYP102 family. Unexpectedly, however, expression of CYP102G4 showed indigo production in LB medium batch flask culture, and high catalytic activity (k_cat/K_m) for indole was measured as 6.14 ± 0.10 min^− 1 mM^− 1. Besides indole, CYP102G4 was able to hydroxylate aromatic compounds such as flavone, benzophenone, and chloroindoles. Homology model has shown such ability to accept aromatic compounds is due to its bigger active site cavity. Unlike other CYP102s, CYP102G4 did not have biased cofactor dependency, which was possibly determined by difference in NAD(P)H binding residues (Ala984, Val990, and Tyr1064) compared to CYP102A1 (Arg966, Lys972 and Trp1046). Overall, a self-sufficient CYP within CYP102G subfamily was characterized using purified enzymes, which appears to possess unique properties such as an only prokaryotic CYP naturally producing indigo.     

Acaricidal activity of Aloe vera L. leaf extracts against Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae)

Four different extracts of Aloe vera L. leaves were evaluated for acaricidal activity against female adults of carmine spider mite, Tetranychus cinnabarinus (Boisduval), by slide-dip bioassay. At 72 h after treatment, the acetone extract showed the strongest acaricidal activity with LC₅₀ value of 90 ppm. The LC₅₀ values for ethyl acetate, water, and ethanol extracts were 113, 340, and 391 ppm, respectively. The acetone extract was fractionated using a silica gel column. Among the twenty-two fractions obtained the fifth, tenth, eleventh, twelfth, fifteenth, and seventeenth fractions showed strong acaricidal activity, causing 80.39 to 92.16% mortality at 72 h after treatment. The tenth and eleventh fractions had the strong activity, with LC₅₀ values of 44 ppm and 33 ppm, respectively. The results suggested that A. vera has a great potential for development as a botanical acaricide for T. cinnabarinus control.