The cruciferous Diplotaxis simplex: Phytochemistry analysis and its protective effect on liver and kidney toxicities, and lipid profile disorders in alloxan-induced diabetic rats.

BACKGROUND: Type 2 diabetes mellitus is a prevalent systemic disease affecting an important proportion of the population worldwide. It has been suggested that excessive reactive oxygen species generation and therefore development of an oxidative stress status is a key factor leading to diabetic complications. Accordingly, it seems that medicinal plants can offer a wide range of new antidiabetic drugs. Diplotaxis simplex (Viv.) Spreng. (Brassicaceae) is an edible plant largely distributed in the Mediterranean region. D. simplex flowers display important in vitro antioxidant potential and inhibitory activity of the α-glucosidase, a key enzyme linked to type 2 diabetes mellitus. In this paper, the antihyperglycemic potential of D. simplex flowers on diabetic rats were investigated. METHODS: Bioactive substances were determined by liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) analysis. Animals were divided into four groups of six rats each: a normal control group, a diabetic control group, a diabetic group receiving flowers extract (200 mg/kg body mass) and a diabetic group receiving acarbose (10 mg/kg body mass) as standard drug. RESULTS: Many glycosides of rhamnetin, isorhamnetin, quercetin and kaempferol compounds were identified in the ethanolic flowers extract. Alloxan induced hyperglycemia, manifested by a significant (p < 0.05) increase in the blood glucose level as well as in serum α-amylase activity. Furthermore, diabetic rats exhibited oxidative stress, as evidenced by a decrease in antioxidant enzymes activities and an increase in lipid peroxidation level of the pancreas, liver and kidneys. Interestingly, the oral administration of D. simplex flowers extract during 30 days restored the glycemia, α-amylase activity, serum lipid profile and antioxidant enzymes. Moreover, the flowers extract exhibited a renal protective role by decreasing the urea and creatinine levels in diabetic rats serum. CONCLUSIONS: D. simplex flowers contained bioactive compounds that possess important antioxidant and hypoglycemic properties and protected pancreas, liver and kidneys against hyperglycemia damage.

Cladodes from prickly pear as a functional ingredient: effect on fat retention, oxidative stability, nutritional and sensory properties of cookies.

The stems of Opuntia ficus-indica known as cladodes are rich source of bioactive and functional substances, which make them important candidate for the production of health-promoting food. Cladodes powder was incorporated at different levels of substitution (2.5%, 5% and 7.5%) in cookies (butter/wheat flour: 55/100 m/m). Substitution of wheat flour by cladodes powder improved dietary fiber, ash, potassium, magnesium and calcium contents of enriched cookies. The results also revealed that cladodes supplementation increased hardness; however, it decreased a* and b* values and reduced exudate loss of cookies during storage. Moreover, rising levels of cladodes powder contribute to the increase of antioxidant activity of cookies and decreased their oxidative degradation. Sensory evaluation showed that cladodes supplementation at 5% level remained acceptable at 5-point hedonic scale. The present study suggested that cladodes supplementation in high-fat cookies not only added nutritional value to food, but also improved its functional characteristics.

Kefir mitigates renal damage caused by zearalenone in female wistar rats by reducing oxidative stress.

The estrogen-like mycotoxin zearalenone (ZEA) was popularly occurred in several food and feeds, posing threats to human and animal health. ZEA induced renal toxicity and caused oxidative stress. In the current study, the protecting effect of kefir administration against ZEA-induced renal damage in rats was explored. Rats were divided into 4 groups, each consisting of 5 animals. For the initial 7 days, they were orally administered sterile milk (200 µL/day). Subsequently, during the second week, the groups were exposed to kefir (200 µL/day), ZEA (40 mg/kg b.w./day) and a combination of kefir and ZEA. The biochemical parameters, kidney histological changes and ZEA residue were assessed. Kefir supplementation enhanced the antioxidant enzymes in the kidney, such as superoxide dismutase, catalase and glutathione peroxidase activities, which increased by 1.2, 4 and 20 folds, respectively, relative to the ZEA group. Remarkably, the concomitant administration kefir + ZEA suppressed ZEA residues in both serum and kidney. Additionally, serum levels of blood urea nitrogen, uric acid and renal malondialdehyde decreased by 22, 65 and 54%, respectively, in the kefir + ZEA group; while, the creatinine content increased by around 60%. Rats co-treated with kefir showed a normal kidney histological architecture contrary to tissues alterations mediated in the ZEA group. These results suggest that kefir may showed a protective effect on the kidneys, mitigating ZEA-induced acute toxicity in rats.

Novel garden cress-fish gelatin based ointment: Improvement of skin wound healing in rats through modulation of anti-inflammatory and antioxidant states.

This study aimed to investigate the ability of aqueous extract of Lepidium sativum seeds (LSE) to improve the wound healing process in rat models. The gelatin, extracted from the skin of smooth-hound shark using citric acid, was used as a support material for ointment. Animals were divided into four groups of six rats each: an untreated control group, a control group treated with Moist Exposed Burn Ointment (MEBO), a treated group with gelatin gel, and a treated group with gelatin gel fortified with 20 mg/mL LSE. Phenolics profile analysis showed that the major compounds in LSE were catechin (125 µg/g) and quinic acid (105 µg/g). In vitro antioxidant tests showed that LSE has interesting activities to scavenge ABTS•+ radicals (IC50 = 0.22 mg/mL) and inhibit the oxidation of linoleic acid. A significant decline in the antioxidant enzymes activities and an increase in the level of thiobarbituric acid reactive substances (TBARS) and inflammatory markers was observed within the injured tissues of the untreated rats compared to rats treated with MEBO. Interestingly, when the wounded tissue was treated with gelatin gel a remarkable reversal of this trend occurred. Further, by enrichment of gelatin gel with LSE, the levels of CAT, GPx and SOD activities significantly increased by 35, 126, and 212 %, respectively, whereas the TBARS level was reduced by 31 %. These results were consistent with the wound contraction percentage and histological analysis, which suggest the potential effect of LSE-enriched gelatin gels to regenerate damaged tissues.

Techno-functional characterization of gelatin extracted from the smooth-hound shark skins: Impact of pretreatments and drying methods.

Gelatin derived from marine by-products could be an interesting alternative to classic mammalian gelatin. The pretreatment and extraction conditions could influence the size of the resulting peptide chains and therefore their techno-functional properties. Thus, it is important to optimize the production process to get a gelatin for the appropriate applications. Skin pretreatment was done by microwaves or oven-drying and the extracted gelatin was dried by spray- or freeze-drying. Freeze-dried gelatin extracted from untreated skin (FGUS) had the highest gelatin yield (10.40%). Gelatin proximate composition showed that proteins were the major component (87.12-89.95%), while lipids showed the lowest contents (0.65-2.26%). Glycine showed the highest level (299-316/1000 residues) in the extracted gelatins. Proline and hydroxyproline residues of gelatins from untreated skin were significantly higher than those from pretreated skin-gelatin. FTIR spectra were characterized by peaks of the amide A (3430-3284 cm-1), B (3000-2931 cm-1), I (1636-1672 cm-1), II (1539-1586 cm-1) and III (1000-1107 cm-1). Spray-drying decreased the gelling properties of gelatins, since it reduced gelling and melting temperatures compared to freeze-drying. Skin pretreatment significantly reduced the gel strength of gelatin by about 50-100 g depending on the gelatin drying method. The FGUS showed better surface properties compared to other gelatins. The highest emulsion activity index (39.42 ± 1.02 m2/g) and foaming expansion (172.33 ± 2.35%) were measured at 3% FGUS. Therefore, the promising properties of freeze-dried gelatin derived from untreated skin, gave it the opportunity to be successfully used as a techno-functional ingredient in many formulations.

Variation of chemical composition of essential oils in wild populations of Thymus algeriensis Boiss. et Reut., a North African endemic species.

UNLABELLED: ABSBACKGROUND: Thymus algeriensis is an endemic aromatic plant to Tunisia largely used in folk medicine and as a culinary herb. The bulks aromatic plants come from wild populations whose essential oils compositions as well as their biological properties are severely affected by the geographical location and the phase of the plant development. Therefore, the aim of the present work is to provide more information on the variation of essential oil composition of T. algeriensis collected during the vegetative and the flowering phases and from eight different geographical regions. Besides, influence of population location and phenological stage on yield and metal chelating activity of essential oils is also assessed. METHODS: The essential oil composition of Thymus algeriensis was determined mainly by GC/FID and GC/MS. The chemical differentiation among populations performed on all compounds was assessed by linear discriminate analysis and cluster analysis based on Euclidean distance. RESULTS: A total of 71 compounds, representing 88.99 to 99.76% of the total oil, were identified. A significant effect of the population location on the chemical composition variability of T. algeriensis oil was observed. Only 18 out of 71 compounds showed a statistically significant variation among population locations and phenological stages. Chemical differentiation among populations was high. Minor compounds play an important role to distinguish between chemical groups. Five chemotypes according to the major compounds have been distinguished. Chemotypes distribution is linked to the population location and not to bioclimate, indicating that local selective environmental factors acted on the chemotype diversity. CONCLUSIONS: The major compounds at the species level were α-pinene (7.41-13.94%), 1,8-cineole (7.55-22.07%), cis-sabinene hydrate (0.10-12.95%), camphor (6.8-19.93%), 4-terpineol (1.55-11.86%), terpenyl acetate (0-14.92%) and viridiflorol (0-11.49%). Based on major compounds, the populations were represented by (α-pinene/1,8-cineole/cis-sabinene hydrate/camphor/viridiflorol), (1,8-cineole/camphor/terpenyl acetate), (α-pinene/1,8-cineole/camphor), (1,8-cineole/camphor/4-terpineol) and (α-pinene/1,8-cineole/cis-sabinene hydrate/camphor/4-terpineol) chemotypes. Variation of phenological stage did not have a statistically significant effect on the yield and metal chelating activity of the essential oil. These results can be used to investigate the geographical location and the harvesting time of this plant for relevant industries.

Development of active edible coatings based on fish gelatin enriched with Moringa oleifera extract: Application in fish (Mustelus mustelus) fillet preservation.

An edible coating was developed using gelatin extracted from the skin of gray triggerfish (Balistes capriscus) and applied to the fillet of the smooth-hound shark (Mustelus mustelus). Moringa oleifera leaf extract was added to gelatin coating solution to improve its preservative properties. The phenolic profiles and antioxidant and antibacterial activities of M. oleifera extracts were determined. Phenolic acids constituted the largest group representing more than 77% of the total compounds identified in the ethanol/water (MOE/W) extract, among which the quinic acid was found to be the major one (31.48 mg/g extract). The MOE/W extract presented the highest DPPH• scavenging activity (IC50 = 0.53 ± 0.02 mg/ml) and reducing (Fe3+) power (EC0.5 = 0.57 ± 0.02 mg/ml), as well as interesting inhibition zones (20-35 mm) for the most tested strains. Coating by 3% of gelatin solution significantly reduced most deterioration indices during chilled storage, such as malondialdehyde (MDA), total volatile basic nitrogen (TVB-N), weight loss, pH, and mesophilic, psychrophilic, lactic, and H2S-producing bacterial counts. Interestingly, coating with gelatin solution containing MOE/W extract at 20 µg/ml was more effective than gelatin applied alone. Compared with the uncoated sample, gelatin-MOE/W coating reduced the weight loss and MDA content by 26% and 70% after 6 days of storage, respectively. Texture analysis showed that the strength of uncoated fillet increased by 46%, while the strength of fillet coated with gelatin-MOE/W only increased by 12% after 6 days of storage. Fish fillet coated with gelatin-MOE/W had the highest sensory scores in terms of odor, color, and overall acceptability throughout the study period.

Blue-green algae (Arthrospira platensis) as an ingredient in pasta: free radical scavenging activity, sensory and cooking characteristics evaluation.

The effects of semolina enrichment with blue-green algae (Arthrospira platensis) at three different levels (1, 2 and 3 g/100 g of semolina) on the colour, cooking properties, firmness, free radical scavenging activity and sensory characteristics of pasta are reported. Microalgae addition resulted in higher swelling index and lower cooking loss than the control sample. A significant increase in pasta firmness was evidenced with an increase of added microalgae due to structural reinforcement. In addition to colouring, the use of A. platensis (2 g/100 g of semolina) can enhance the sensory quality and nutraceutical potential as evaluated by free radical scavenging activity of pasta.

Volatile and lipid analyses by gas chromatography/mass spectrometry and nutraceutical potential of edible wild Malva aegyptiaca L. (Malvaceae).

Volatile and lipid chemical compositions, and nutritional and antioxidant properties of Malva aegyptiaca, an edible wild plant largely distributed in North Africa, were investigated. Forty-nine compounds of volatiles were identified showing large qualitative and quantitative differences during three phenological stages. The flowering stage was characterized by the presence of a high number of terpenic compounds, among them dillapiole was found to be the major one (55.15%). The nutrient composition of leaves and fruits was investigated in the present work. Fruits' lipidic fraction was characterized by its high level of linoleic acid (n-6) (36.17%). Interestingly, leaves' lipidic fraction was characterized by its very high content of camphor (43.69%) and by its relatively high content of linoleinic acid (n-3) (14.69%). Furthermore, our results showed that the phenolic contents varied from 352 to 404 mg gallic acid equivalent/g ethanolic and acetonic extracts, respectively. These extracts revealed interesting antioxidant activities including free radical scavenging activity (EC(50) = 0.38-0.57 mg/ml) and reducing power (EC(50) = 0.12-0.18 mg/ml).

Development and characterization of fish gelatin-based biodegradable film enriched with Lepidium sativum extract as active packaging for cheese preservation.

The physical and functional properties of gelatin-based films enriched with organic extracts from Lepidium sativum seeds were studied. Gelatin was extracted from the skin of dogfish (Squalus acanthias) and the functional gelatin-based films were used to preserve cheese during chilled storage. Ethanol extract (LSE3) and gelatin-based film enriched with LSE3 at 20 µg/mL showed high antioxidant potential using various complementary methods. No significant difference was measured in the mechanical parameters of the enriched films in terms of thickness, tensile strength and elongation at break. LSE3 incorporation at the highest level slighltly decreased the film L∗ value from 90.30 ± 0.10 to 88.10 ± 0.12, while the b∗ value increased from 0.91 ± 0.07 to 8.89 ± 0.12. Wrapping the cheese with gelatin-based film enriched with 20 µg LSE3/mL reduced the syneresis by 40% and stabilized the color, peroxidation and bacteria growth as compared to the unwrapped sample after 6 days of storage. In addition, cheese wrapped with the active gelatin-based film showed the lowest changes in texture parameters. Overall results suggest the use of the enriched gelatin film as active packaging material to preserve cheese quality.

Chicken feathers: a complex substrate for the co-production of alpha-amylase and proteases by B. licheniformis NH1.

This study is concerned with the co-production of alkaline proteases and thermostable alpha-amylase by some feather-degrading Bacillus strains: B. mojavensis A21, B. licheniformis NH1, B. subtilis A26, B. amyloliquefaciens An6 and B. pumilus A1. All strains produced both enzymes, except B. pumilus A1, which did not exhibit amylolytic activity. The best enzyme co-production was obtained by the NH1 strain when chicken feathers were used as nitrogen and carbon sources in the fermentation medium. The higher co-production of both enzymes by B. licheniformis NH1 strain was achieved in the presence of 7.5 g/l chicken feathers and 1 g/l yeast extract. Strong catabolic repression on protease and alpha-amylase production was observed with glucose. Addition of 0.5% glucose to the feather medium suppressed enzyme production by B. licheniformis NH1. The growth of B. licheniformis NH1 using chicken feathers as nitrogen and carbon sources resulted in its complete degradation after 24 h of incubation at 37 degrees C. However, maximum protease and amylase activities were attained after 30 h and 48 h, respectively. Proteolytic activity profiles of NH1 enzymatic preparation grown on chicken feather or casein-based medium are different. As far as we know, this is the first contribution towards the co-production of alpha-amylase and proteases using keratinous waste. Strain NH1 shows potential use for biotechnological processes involving keratin hydrolysis and industrial alpha-amylase and proteases co-production. Thus, the utilization of chicken feathers may result in a cost-effective process suitable for large-scale production.

Microstructure and characteristic properties of dogfish skin gelatin gels prepared by freeze/spray-drying methods.

The effects of two pretreatments (microwaves or oven-drying) on the dogfish (Squalus acanthias) skin as well as two drying processes (freeze-drying or spray-drying) on the extracted gelatins were studied. Thus six types of gelatins were obtained, three of which were freeze-dried (FG) and the others were spray-dried (SG), from the untreated skin (US), microwaves-pretreated skin (MS) and oven-pretreated skin (OS). The highest yield (8.67%) was obtained for the OSFG, while the lowest one (3.06%) was measured for the OSSG. Interestingly, all gelatins exhibited relatively high protein (84.02-89.53%), and low lipid (0.50-1.71%) and ash (3.05-7.17%) contents. In addition, gelatins were analyzed by the Fourier transform infrared and the spectra displayed important differences in some specific peaks, particularly in the amide I, amide II and amide III. The gelatins extracted from the untreated skin, regardless the drying method, presented the highest foaming capacity. The textural profile analysis showed that USSG was the hardest (213.6 g) and the chewier (23.8 N × mm) gelatin. Moreover, analysis of thermal properties showed that USSG also has the highest glass-transition temperature. The interesting properties of gelatin extracted from dogfish skin encourage their future use as a functional ingredient in industrial food formulations.

Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing.

The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses.

Isolation of polysaccharides from Malva aegyptiaca and evaluation of their antioxidant and antibacterial properties.

This study investigated the antioxidant and antibacterial properties of crude water-soluble polysaccharides extracted from the mallow (Malva aegyptiaca) by precipitation with cetylpyridinium chloride (P1) or ethanol (P2). The Polysaccharides fractions were characterized by FTIR spectroscopy, a monosaccharide composition, and antioxidant and antibacterial activities. P1 showed the highest total sugars (81.2%) and sulfated groups (2.9%) contents but with the lowest total proteins content (8.7%). The infrared spectroscopic spectra exhibited the typical bands and peak characteristic of polysaccharides. Monosaccharides analysis revealed the dominance of galactose (40.3-43.7%) and glucuronic acid (25.9-30.9%). Interestingly, P1 displayed an important antioxidant activity as evaluated by the (Fe2+) chelating activity (IC50=1.15mg/ml), (Fe3+) reducing power (EC50=1.22mg/ml), ß-carotene bleaching inhibition capacity (IC50=1.56mg/ml) and DPPH-radical scavenging activity (IC50=1.94mg/ml). Furthermore, P1 at 10mg/ml was highly active than P2 against several bacterial strains and especially the Gram-positive bacteria, where the inhibition rate ranged between 84.2 and 90.3%. Obtained results suggest that M. aegyptiaca polysaccharides precipitated by cetylpyridinium chloride could be used as a naural antioxidant and antibacterial agent.

Essential oil variation in wild populations of Artemisia saharae (Asteraceae) from Tunisia: chemical composition, antibacterial and antioxidant properties.

BACKGROUND: Artemisia saharae Pomel is a new taxon of Artemisia herba-alba Asso (Asteraceae) which is endemic to Tunisia and Algeria. This shrub, commonly known as white wormwood or desert wormwood, is largely used in folk medicine and as a culinary herb. The bulks aromatic plants come from wild populations whose essential oils compositions as well as their biological properties are severely affected by several factors such as geographic conditions. Therefore, the aim of the present work is to provide more information about the influence of altitude variation on the essential oil composition, antimicrobial and antioxidant properties of Artemisia saharae growing wild in the same geographical area. RESULTS: Essential oils were extracted by hydrodistillation of leaves and flowers of the plant collected from seven different altitudes of the Baten Zamour region (southwest of Tunisia). The highest essential oil yields (2.70-2.80%) were obtained for populations of high altitudes. Seventy-five compounds, representing 92.78 to 96.95% of the total essential oils, were separated and identified. Essential oils were characterized by very high percentage of oxygenated monoterpenes (52.1-72.6%) which constituted the predominant class. From the analyzed populations, the major compounds (>7%) were α-thujone, ß-thujone, chrysanthenone, camphor, chrysanthenyl acetate, and sabinyl acetate. Sabinyl acetate which was detected in some populations at relatively high percentages (7.7-10.8%) seems to be characteristic to Southern Tunisian A. saharae. The studied essential oil showed a chemical diversity depending on the population altitude as revealed by linear discriminant and cluster analyses. CONCLUSIONS: Three population groups associated with altitudinal levels were distinguished. It is worthy to note that the most discriminating compounds of chemical groups were the minor ones. Despite the high variation of essential oil compositions, the high altitude population did not affect severely the antibacterial activity against the most tested strains. Altitude seems to be an important factor influencing the yield and the chemical profile of Artemisia saharae essential oils. Knowledge of the chemical composition of essential oils in relation to environmental factors is a very important quality criterion for their marketing and contributes to their valorization as functional ingredient in food technology.

Alkaline chymotrypsin from striped seabream (Lithognathus mormyrus) viscera: purification and characterization.

An alkaline chymotrypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified by precipitation with ammonium sulfate, Sephadex G-100 gel filtration, Mono Q-Sepharose anion-exchange chromatography, ultrafiltration, second Sephadex G-100 gel filtration, and a second Mono Q-Sepharose anion-exchange chromatography with a 80-fold increase in specific activity. The molecular weight of the purified alkaline chymotrypsin was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 10.0-11.0 using succinyl-L-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPNA) as a substrate. The relative activities at pH 7.0 and 12.0 were about 66% and 45.5%, respectively. Further, the enzyme was extremely stable over a broad pH range (6.0-12.0). The optimum temperature for enzyme activity was 50 degrees C, and the enzyme displayed higher enzyme activity at low temperatures when compared to other enzymes. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulfonyl-fluoride (PMSF), a serine protein inhibitor, and N-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), a chymotrypsin specific inhibitor. The N-terminal amino acid sequence of the first nine amino acids was IVNGEEAVP. The chymotrypsin kinetic constants, Km and kcat on SAAPNA as a substrate, were 30.7 microM and 14.35 s(-1), respectively, while the catalytic efficiency kcat/Km was 0.465 microM(-1) s(-1). The high activity at high alkaline pH and low temperatures make this protease a potential candidate for future use in detergent processing industries.

Production and biochemical and molecular characterization of a keratinolytic serine protease from chicken feather-degrading Bacillus licheniformis RPk.

A novel feather-degrading bacterium was isolated from a polluted river and identified as Bacillus licheniformis RPk. The isolate exhibited high proteinase production when grown in chicken-feather media. Complete feather degradation was achieved during cultivation. Maximum protease activity (4150 U/mL with casein as a substrate and 37.35 U/mL with keratin as a substrate) was obtained when the strain was grown in a medium containing 7.5 g/L chicken feathers, 2 g/L yeast extract, 0.5 g/L NaCl, 0.1 g/L MgSO4 x 7H2O, 0.7 g/L KH2PO4, and 1.4 g/L K2HPO4 for 48 h with agitation of 200 rev/min at 37 degrees C. The major protease produced by B. licheniformis RPk was purified to homogeneity by a 3-step procedure. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE and gel filtration. The optimum pH and temperature for the caseinolytic activity were around 11.0 and 65 degrees C, respectively. The optimum pH and temperature for the keratinolytic activity were 9.0 and 60 degrees C, respectively. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, which suggests that the purified enzyme is a serine protease. The thermostability of the enzyme was considerably enhanced in the presence of Ca2+ at temperatures >50 degrees C. The kerRP gene, which encodes the keratinolytic protease, was isolated, and its DNA sequence was determined. The deduced amino acid sequence revealed that the keratinase KerRP differs from KerA of B. licheniformis PWD-1, subtilisin Carlsberg, and keratinase of B. licheniformis by 2, 4, and 62 amino acids, respectively.