An antifungal chitinase produced by Bacillus cereus with shrimp and crab shell powder as a carbon source

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish processing wastes. In this study, shrimp and crab shell powder prepared by treating shrimp and crab processing wastes with boiling and crushing was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus cereus YQ 308, a strain isolated from the soil samples, excreted one chitinase when cultured in a medium containing 2% (wt/vol) shrimp and crab shell powder as major carbon source. The chitinase, purified by sequential chromatography, had an Mr of 48 kDa and pI of 5.2. The purified chitinase (2 mg/ml) inhibited the hyphal extension of the fungi Fusarium oxysporum and Pythium ultimum.     

An Antifungal Chitinase Produced by <Emphasis Type="Italic">Bacillus cereus</Emphasis> with Shrimp and Crab Shell Powder as a Carbon Source

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish processing wastes. In this study, shrimp and crab shell powder prepared by treating shrimp and crab processing wastes with boiling and crushing was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus cereus YQ 308, a strain isolated from the soil samples, excreted one chitinase when cultured in a medium containing 2% (wt/vol) shrimp and crab shell powder as major carbon source. The chitinase, purified by sequential chromatography, had an Mr of 48 kDa and pI of 5.2. The purified chitinase (2 mg/ml) inhibited the hyphal extension of the fungi Fusarium oxysporum and Pythium ultimum. RID= ID= <E5>Correspondence to: </E5>S.-L. Wang; <E5>email:</E5> sabulo&commat;mail.dyu.edu.tw Received: 27 August 2002 / Accepted: 25 September 2002     

Proficient biodegradation of shrimp shell waste by Aeromonas hydrophila SBK1 for the concomitant production of antifungal chitinase and antioxidant chitosaccharides

This study aimed to optimize the biodegradation of shrimp shell waste by Aeromonas hydrophila SBK1 for the co-production of chitinase and chitosaccharides (CS) under submerged fermentation and evaluation of their bioactivities. Canonical analysis and parametric optimization wrought the peakest production of chitinase (21.48 U/ml) and CS (124 μg/ml) after 66.4 h of fermentation at 37.6 °C. The medium containing 2.64% (w/v) shrimp shell powder, 0.38% (w/v) NaCl, 6.86 × 10⁶ cfu/ml inoculum concentration and an agitation speed of 120 rpm were found best. These optimized parameters were also authenticated by scale up of fermentation in 5 L fermentor and a reproducible results obtained with specific yield of chitinase (Y_P/S^chi) of 958.82 U/g and CS (Y_P/S^CS) 5.5 mg/g. A 59 kD chitinase was purified from culture filtrate by sequential chromatography techniques. The enzyme exhibited high degree of antifungal activity particularly against pathogenic Aspergillus flavus and Fusarium oxysporum by dissolving their cell wall components. The IC₅₀ values for A. flavus and F. oxysporum were 3.7 and 4.5 U/ml of purified chitinase, respectively. Chitosaccharides were extracted from the culture filtrate, quantitatively identified as admixture of N-acetylglucosamine monomer (57.5%) and dimer (39.2%). These chitosaccharides have potential antioxidant activity as detected by in vitro free radical scavenging assay.     

Extraction,purification and characterization of an antioxidant from marine waste using protease and chitinase cocktail

Marine waste is a highly renewable resource for the recovery of several value added metabolites with prospective industrial applications. This study describes the production of enzymes on marine waste and their subsequent use for the extraction of antioxidants from marine waste. Microbispora sp. and Bacillus sp. were grown on colloidal chitin and marine waste for the production of chitinase and protease. Microbispora sp. could produce 10.2 U ml⁻¹ chitinase, whereas Bacillus sp. could produce 38 U ml⁻¹ chitinase and 3.39 U ml⁻¹ protease. The production of antioxidants was optimized using statistical designs and 6.6 units of 35 kDa chitinase from Microbispora sp., 16 units of 25 kDa chitinase from Bacillus sp., 2.3 units of protease, 1.5% marine waste and 36 h incubation gave maximum antioxidant activity. Nearly 5.0 mg of compound with antioxidant activity could be recovered per gram of marine waste. This compound was purified by HPLC and characterized by TLC, FT-IR and proton-NMR as N,N′-diacetylchitobiose. It exhibited 53% superoxide radical scavenging activity, 57% hydroxyl radical scavenging activity and 28% lipid peroxidation inhibition activity. Scale up of the extraction of antioxidant from marine waste and its pharmacological studies can extend its use in medicine.     

Production of Antifungal Chitinase by Aspergillus niger LOCK 62 and Its Potential Role in the Biological Control

Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6?days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43?kDa. The highest activity was obtained at 40?°C for both crude and purified enzymes. The crude chitinase activity was stable during 180?min incubation at 40?°C, but purified chitinase lost about 25?% of its activity under these conditions. Optimal pH for chitinase activity was pH 6–6.5. The activity of crude and purified enzyme was stabilized by Mg²⁺ and Ca²⁺ ions, but inhibited by Hg²⁺ and Pb²⁺ ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected.     

Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium

A fungus with protease and chitinase activities was isolated from the soil. It has been identified as Aspergillus fumigatus Fresenius TKU003. A. fumigatus TKU003 produced proteases and chitinases when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine waste. An extracellular protease was purified from the culture supernatant of A. fumigatus TKU003. The molecular weight of TKU003 protease was 124 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pI for TKU003 protease was 8.3. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU003 protease was pH 8, 40 °C, 6–10, and 50 °C, respectively. The activity of the enzyme was strongly inhibited by PMSF. TKU003 serine protease, same as most other serine proteases of A. fumigatus, belongs to protease with alkaline pI. The unique characteristics of TKU003 protease is its high molecular weight.     

Characterization of Antifungal Chitinase from Marine Streptomyces sp. DA11 Associated with South China Sea Sponge Craniella Australiensis

The gene cloning, purification, properties, kinetics, and antifungal activity of chitinase from marine Streptomyces sp. DA11 associated with South China sponge Craniella australiensis were investigated. Alignment analysis of the amino acid sequence deduced from the cloned conserved 451 bp DNA sequence shows the chitinase belongs to ChiC type with 80% similarity to chitinase C precursor from Streptomyces peucetius. Through purification by 80% ammonium sulfate, affinity binding to chitin and diethylaminoethyl-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg⁻¹ was achieved. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular weight of approximately 34 kDa and antifungal activities were observed against Aspergillus niger and Candida albicans. The optimal pH, temperature, and salinity for chitinase activity were 8.0, 50°C, and 45 g‰ psu, respectively, which may contribute to special application of this marine microbe-derived chitinase compared with terrestrial chitinases. The chitinase activity was increased by Mn²⁺, Cu²⁺, and Mg²⁺, while strongly inhibited by Fe²⁺ and Ba²⁺. Meanwhile, SDS, ethyleneglycoltetraacetic acid, urea, and ethylenediaminetetraacetic acid were found to have significantly inhibitory effect on chitinase activity. With colloidal chitin as substrates instead of powder chitin, higher V _max (0.82 mg product/min·mg protein) and lower K _m (0.019 mg/ml) values were achieved. The sponge’s microbial symbiont with chitinase activity may contribute to chitin degradation and antifungal defense. To our knowledge, it was the first time to study sponge-associated microbial chitinase.     

Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes

Bacillus cereus QQ308 produced antifungal hydrolytic enzymes, comprising chitinase, chitosanase and protease, when grown in a medium containing shrimp and crab shell powder (SCSP) produced from marine waste. The growth of the plant-pathogenic fungi Fusarium oxysporum, Fusarium solani, and Pythium ultimum were considerably affected by the presence of the QQ308 culture supernatant. The supernatant inhibited spore germination and germ tube elongation of F. oxysporum, F. solani, and P. ultimum. The increase in the growth time of the fungal culture was associated with a gradual decrease in inhibition. Besides antifungal activity, QQ308 enhanced growth of Chinese cabbage. These characteristics were unique among known strains of B. cereus. To our knowledge, this is the first report on the antifungal and Chinese cabbage growth enhancing compounds produced by B. cereus.     

Solid-state cultivation of Bacillus thuringiensis R 176 with shrimp shells and rice straw as a substrate for chitinase production

In this study, shrimp shell powder, prepared by treating shrimp-processing waste by boiling and crushing, was used as a substrate for isolation of chitinase-producing microorganism. These organisms may have an important economic role in the biological control of rice and other fungal pathogens. Two hundred strains of bacteria with the ability to degrade chitin from shrimp shell waste were isolated from paddy soil, and of these, 40 strains showed chitinase activity in a solid state cultivation. One of the most potent isolates (strain R 176) was identified as Bacillus thuringiensis. Identification was carried out using morphological and biochemical properties along with 16S rRNA sequence analysis. This strain was able to produce high levels of extracellular chitinase in solid media containing shrimp shells as sole carbon source [1.36 U/g initial dry substrate (IDS)], which was 0.36-fold higher than the productivity in a liquid culture with colloidal chitin. The effects of medium composition and physical parameters on chitinase production by this organism were studied. The optimal medium contained shrimp shell mixed with rice straw in 1:1 ratio added with ball-milled chitin 0.5 % (w/v) and ammonium sulfate 0.5 % (w/v). The highest enzyme production (3.86 U/g IDS) by B. thuringiensis R 176 was obtained at pH 7, 37 °C after 14 days growth. With respect to the high amount of chitinase production by this strain in a simple medium, this strain could be a suitable candidate for the production of chitinase from chitinous solid substrates, and further investigations into its structure and characteristics are merited.     

Efficient decomposition of shrimp shell waste using <Emphasis Type="Italic">Bacillus cereus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis>

Two bacterial cultures were isolated and tested for degradation of shrimp shell waste. According to morphological examination, physiological tests, and applied molecular techniques, isolates were identified as Bacillus cereus and Exiguobacterium acetylicum. Both strains were cultivated separately in flasks with 100 mL of shrimp shell waste broth (3% of washed, dried and ground shrimp shell waste in tap water, pH 7.0) at 37°C. At determined periods of time, deproteinization and demineralization of residuals were measured. Fermentation of 3% shell waste with B. cereus indicated 97.1% deproteinization and 95% demineralization. For E. acetylicum, the level of deproteinization and demineralization was 92.8 and 92%, respectively. Protein content was reduced from 18.7 to 5.3% with B. cereus and to 7.3% with E. acetylicum. No additional supplements were used during the fermentation of shell waste. B. cereus strain showed higher efficacy in decomposition of shell waste and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth. Incubation of bacteria with shell waste during 14 days at 37°C resulted in 78.6% deproteinization and 73% demineralization. High activity of isolated cultures in decomposition of shrimp shell waste suggests broad potential for application of these bacteria in environmentally friendly approaches to chitin extraction from chitin-rich wastes.     

Marine ash‐products influence growth and feed utilization when Atlantic cod Gadus morhua L. are fed plant‐based diets

Atlantic cod Gadus morhua L. were fed high plant protein diets added to either shrimp‐shell meal or crab‐shell meal. The aims were to investigate if diluting dietary energy would reduce the liver index (HSI) and if marine ash would add value to plant protein‐based diets. Two control diets were used: a high plant protein control diet (PP) with no marine ash addition, and a fishmeal‐based diet (FM) with no marine ash addition. All diets were evaluated in small cod (initial weight 79 ± 15g) and in market‐size cod (initial weight 1579 ± 20 g). Addition of crab‐shell meal up to 20% and shrimp‐shell meal up to 10% did not influence liver size in either small or market‐size cod. An addition of up to 20% crab‐shell meal and 10% shrimp‐shell meal improved growth compared to the PP control diet, and stimulated increased feed intake. However, 10% shrimp‐shell meal and 20% crab‐shell meal diets resulted in a similar intake of energy and protein as the control groups. Increasing shrimp‐shell meal to 20% resulted in reductions in feed intake, fat digestibility and growth, and in altered gut histology. All diets, except the 20% shrimp added diet, resulted in normal ranges of plasma nutrients and blood hematological values, showing good fish health with or without the marine ash addition.     

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)₂) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)_n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)_4–6 and produced (GlcNAc)_2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)₅ to produce (GlcNAc)₂ (79.2%) and (GlcNAc)₃ (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)_n (n = 2–4) was pNp-(GlcNAc)₂ >> pNp-(GlcNAc)₄ > pNp-(GlcNAc)₃. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)_n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.     

Purification and characterization of solvent stable,alkaline protease from Bacillus firmus CAS 7 by microbial conversion of marine wastes and molecular mechanism underlying solvent stability

A marine bacterium Bacillus firmus CAS 7 produced protease in the medium supplemented with 3:1 shrimp and crab shell powder at 55 °C and was purified with the specific activity of 473.4 U/mg. The purified protease was highly stable up to 70 °C, pH 11.0 and 30% NaCl. The protease purified was quite stable in the presence of anionic and non-ionic surfactants and organic solvents. The molecular dynamics simulation confirmed that the competition between organic solvent and water for the enzyme surface was comparatively higher in water–miscible organic solvent which is responsible for organic solvent stability. The purified protease from B. firmus CAS 7 could be greatly useful to develop industrial processes performed under harsh conditions or with denaturants and organic solvents. The protease production by microbial conversion of marine wastes suggested its potential utilization to generate high value-added products.     

Isolation of <Emphasis Type="Italic">Serratia marcescens</Emphasis> SR1 as a Source of Chitinase Having Potentiality of Using as a Biocontrol Agent

Serratia marcescens, strain SR₁ was isolated from the local soil of a cultivated farm and it was screened as potent strain for chitinase production. Maximum chitinase production (77.3 u Mh⁻¹ 100⁻¹) was observed after 96 h of incubation period with pH 5.5 at 30°C under shake conditions (120 rpm). Compare to still flasks, shake culture with prawn fish colloidal chitin of 0.5% (w/v) concentration, showed a better enzyme yield. Crude enzyme showed antifungal activity against plant pathogens.     

High production of cold-tolerant chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus Lecanicillium muscarium CCFEE 5003: Bioprocess optimization and characterization of two main enzymes

The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flasks to check its chitinase production on rough shrimp and crab wastes. Production on shrimp shells was much higher than that on crab shells (104.6 ± 9.3 and 48.6 ± 3.1 U/L, respectively). For possible industrial applications, bioprocess optimization was studied on shrimp shells in bioreactor using RSM to state best conditions of pH and substrate concentration. Optimization improved the production by 137% (243.6 ± 17.3). Two chitinolytic enzymes (CHI1 and CHI2) were purified and characterized. CHI1 (MW ca. 61 kDa) showed optima at pH 5.5 and 45 °C while CHI2 (MW ca. 25 kDa) optima were at pH 4.5 and 40 °C. Both enzymes maintained high activity levels at 5 °C and were inhibited by Fe⁺⁺, Hg⁺⁺ and Cu⁺⁺. CHI2 was strongly allosamidin-sensitive. Both proteins were N-acetyl-hexosaminidases (E.C. 3.2.1.52) but showed different roles in chitin hydrolysis: CHI1 could be defined as “chitobiase” while CHI2 revealed a main “eso-chitinase” activity.     

Occurrence and Activity of Microorganisms in Shrimp Waste

This study aimed to determine the occurrence and respiration activity of heterotrophic bacteria and fungi in shrimp shell waste and to evaluate the role of chitinolytic bacteria and fungi in its decomposition. The highest levels of bacteria were found in shrimp heads sections and the lowest in exoskeletons. The level of fungi was much lower, with the highest proportion present in heads sections and the lowest in exoskeletons. Chitinolytic bacteria constituted a small percentage of the total heterotrophic bacteria in fresh shrimp waste, averaging 4% in exoskeletons, 2.4% in all parts, and 2% in heads. No chitinolytic bacteria were detected in stored waste. In contrast, the percentage of chitinolytic fungi in shrimp waste was much higher than that of bacteria. Chitinolytic fungi constituted 25–60% of the total fungi in fresh waste and 15–40% in stored waste. Chitinolytic bacteria isolated from heads sections were characterized by the highest chitinolytic activity, averaging 11.2 nmol of methylumbelliferyl · mg⁻¹ protein · h⁻¹, whereas the lowest activity was in strains from exoskeletons, averaging 3.2 nmol of methylumbelliferyl · mg⁻¹ protein · h⁻¹. The chitinolytic activity of fungi isolated from all parts waste, head sections, and exoskeletons was similar. The respiration activity of microorganisms in fresh and stored waste was similar. Oxygen consumption activity increased during incubation and approached a saturation value between days 4 and 5. No correlation between the end value of respiratory activity in the analyzed section of shrimp discard after 5 days and the level of bacteria and fungi was observed. The only significant correlation observed was between the respiratory activity of the shrimp and the level of fungi. The respiration activity significantly depended on the analyzed section of shrimp discard (p < 0.000).     

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.     

Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation

A highly chitinolytic strain Penicillium ochrochloron MTCC 517 was procured from MTCC, Chandigarh, India. Culture medium supplemented with 1% chitin was found to be suitable for maximum production of chitinase. Purification of extracellular chitinase was done from the culture medium by organic solvent precipitation and DEAE-cellulose column chromatography. The chitinase was purified 6.92-fold with 29.9% yield. Molecular mass of purified chitinase was found to be 64 kDa by SDS-PAGE. The chitinase showed optimum temperature 40 °C and pH 7.0. The enzyme activity was completely inhibited by Hg²⁺, Zn²⁺, K⁺ and NH₄⁺. The enzyme kinetic study of purified chitinase revealed the following characteristics, such as apparent K_m 1.3 mg ml^?1, V_max 5.523 × 10^?5 moles l^?1 min^?1 and K_cat 2.37 s^?1 and catalytic efficiency 1.82 s^?1 M^?1. The enzyme hydrolyzed colloidal chitin, glycol chitin, chitosan, glycol chitosan, N,N′-diacetylchitobiose, p-nitrophenyl N-acetyl-β-d-glucosaminide and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. The chitinase of P. ochrochloron MTCC 517 is an exoenzyme, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin. Protoplasts with high regeneration capacity were obtained from Aspergillus niger using chitinase from P. ochrochloron MTCC 517. Since it also showed antifungal activity, P. ochrochloron MTCC 517 seems to be a promising biocontrol agent.     

Characterization of chitinase from Streptomyces luridiscabiei U05 and its antagonist potential against fungal plant pathogens

Streptomyces luridiscabiei U05 was isolated from wheat rhizosphere. It produced chitinase, which showed in vitro antifungal properties. The crude enzyme inhibited the growth of Alternaria alternata, Fusarium oxysporum, F. solani, Botrytis cinerea, F. culmorum and Penicillium verrucosum. The chitinase enzyme of the molecular weight of 45 kDa was purified using affinity chromatography of chitin. Streptomyces luridiscabiei U05 produced different chitinolytic enzymes. The highest enzyme activity was observed with the use of 4‐MU‐(GlcNAc)_, which points to the presence of an β‐N‐acetylhexosaminidase. The optimum activity was obtained at 35–40°C and pH 7–8. The enzyme showed thermostability at 35–40°C during 240 min of preincubation and lost its activity at 50°C and 60°C in 60 min. The chitinase activity from S. luridiscabei U05 was strongly inhibited by Hg²⁺ and Pb²⁺ ions, and sodium dodecyl sulphate (SDS). The Ca²⁺, Cu²⁺ and Mg²⁺ ions stimulated the activity of the enzyme.     

Purification and characterization of a protease extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium

Monascus purpureus CCRC31499 produced a protease when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine wastes. An extracellular protease was purified from the culture supernatant to homology. The protease had a molecular weight of 40,000 and a pI of 7.9. The optimal pH, optimum temperature, pH stability, and thermal stability of the protease were pH 7–9, 40 °C, pH 5–9, and 40 °C, respectively. In addition to protease activity, CCRC31499 also exhibited activity of enhancing vegetable growth in culture supernatant. This is also the first report of isolation of a protease from Monascus species.