Keratinase Research Articles

Production, purification and characterization of a proteolytic enzyme from Streptomyces sp. 2M21

The recent application studies on keratinase lead to new application areas for the enzyme to be used in novel industries such as pharmaceuticals and biodegradable composites. Thus, designing an economical and environmentally friendly keratinase production and purification with new features turned into an intriguing objective for its commercialization. In this study, keratinase enzyme was produced using a bioreactor of 5 L by a native Streptomyces sp. 2M21 and purified by ion exchange chromatography using step-wise gradient with 24.1-fold purification. Purified keratinase showed optimum activity at pH 9 and 30 °C. The enzyme was shown to be able to decompose different complex substrates such as chicken feather, wool and nail. Moreover, the addition of DTT enhanced the hydrolysis of wool and chicken feather to 3.4- and 3.2-fold, respectively. The potential of Streptomyces sp. to produce significant levels of keratinase using feathers as a substrate might establish cleaner applications for the production of high value-added products and degrading of keratin-containing wastes.

Optimization of azo-keratin hydrolysis by alginate-immobilized Keratinase produced from Bacillus licheniforms

Recently, much attention has been paid to Keratinolytic materials which can be converted into feed-stuffs, biofertilizers, glues, and foils or used for production of amino acids. Here, we determined the optimum conditions for keratin hydrolysis by alginate-immobilization of crude keratinase isolated from Bacillus lichenoformis. The results of this study indicated that the optimum pH for keratin degradation by immobilized keratinase enzyme was pH 8. Moreover, the crude enzyme was stable for 2 hours at pH 4, 7 and 10 and retained 55.5%, 60% and 53%, of its activity respectively for immobilized enzyme while free enzyme retained only 40%, 40% and 61%, respectively. The optimum temperature for keratinase activity was 50 oC when immobilized in alginate. At temperatures 30, 45 and 60 oC, keratinase was stable for 2 hours and retained 57%, 66% and 57%, of its activity respectively for immobilized enzyme while free enzyme retained only 51%, 62% and 51%, respectively. Furthermore, the effects of activators and inhibitors on the enzyme activity have been investigated.

Effective production of keratinase by gellan gum-immobilised Alcaligenes sp. AQ05-001 using heavy metal-free and polluted feather wastes as substrates.

The ability of gellan gum-immobilised cells of the heavy metal-tolerant bacterium Alcaligenes sp. AQ05-001 to utilise both heavy metal-free and heavy metal-polluted feathers (HMPFs) as substrates to produce keratinase enzyme was studied. Optimisation of the media pH, incubation temperature and immobilisation parameters (bead size, bead number, gellan gum concentration) was determined for the best possible production of keratinase using the one-factor-at-a-time technique. The results showed that the immobilised cells could tolerate a broader range of heavy metal concentrations and produced higher keratinase activity at a gellan gum concentration of 0.8% (w/v), a bead size of 3mm, bead number of 250, pH of 8 and temperature of 30°C. The entrapped bacterium was used repeatedly for ten cycles to produce keratinase using feathers polluted with 25ppm of Co, Cu and Ag as substrates without the need for desorption. However, its inability to tolerate/utilise feathers polluted with Hg, Pb, and Zn above 5ppm, and Ag and Cd above 10ppm resulted in a considerable decrease in keratinase production. Furthermore, the immobilised cells could retain approximately 95% of their keratinase production capacity when 5ppm of Co, Cu, and Ag, and 10ppm of As and Cd were used to pollute feathers. When the feathers containing a mixture of Ag, Co, and Cu at 25ppm each and Hg, Ni, Pb, and Zn at 5ppm each were used as substrates, the immobilised cells maintained their operational stability and biological activity (keratinase production) at the end of 3rd and 4th cycles, respectively. The study indicates that HMPF can be effectively utilised as a substrate by the immobilised-cell system of Alcaligenes sp. AQ05-001 for the semi-continuous production of keratinase enzyme.

Feather degradation by keratinolytic bacteria and biofertilizing potential for sustainable agricultural production.

Feathers account for 5-7% of the total weight of chicken have become one of the major pollutants due to their recalcitrant nature. Feather which is constituted of 90% keratin can be a good source of peptides, amino acids, and minerals for use as organic fertilizer. Traditional feather degradation methods consume large amount of energy and reduces the overall quality of the proteins. However, degradation of keratin by keratinolytic bacteria may represent as an alternative for the development of cheap, cost effective, eco-friendly, and easily available nitrogen (N) and minerals rich source as potential organic fertilizers. Keratinase enzymes from bacteria are serine-type proteases showing optimal activity at pH 6 to 9 and 30 to 50 °C. Mechanism of degradation includes, sulfitolysis, proteolysis, followed by deamination. Keratinolytic bacteria showing antagonism against important plant pathogens may act as biocontrol agent. Feather hydrolyzate can also be employed as nitrogenous fertilizers for plant growth. Tryptophan release from the feather degradation can act as precursor for plant phytohormone, indole-3-acetic acid (IAA). Solubilization of inorganic phosphate (P) by keratinolytic bacteria may further elevate the growth of plant. Application of hydrolyzate increases the water holding capacity, N, carbon (C) and mineral content of the soil. It elevates protein, amino acids, and chlorophyll content of plant. Feather hydrolyzate enhances seed germination and growth of plant. Soil application further increases the population of beneficial bacteria. The use of keratinolytic bacteria having antagonistic and plant growth promoting activities, and feather hydrolyzate can emerge as sustainable and alternative tools to promote and improve organic farming, agro-ecosystem, environment, human health, and soil biological activities.

Effect of keratinase on ileal amino acid digestibility in five feedstuffs fed to growing pigs

ObjectiveThis study was conducted to evaluate the effect of keratinase (KE) on the apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of amino acids (AA) in rice bran, cottonseed meal (CSM), rapeseed meal (RSM), corn distillers dried grains with solubles (DDGS), and peanut meal (PNM).MethodsTwelve crossbred barrows (Duroc×Landrace×Yorkshire, 50.5±1.4 kg body weight [BW]) fitted with T-cannulas at the terminal ileum were allotted to a 12×6 Youden Square design with 12 diets and 6 periods. The treatment diets included rice bran, CSM, RSM, corn DDGS, PNM, or corn-soybean meal (cSBM) supplemented with 0.05% KE or not. Diets were given to pigs at a level of 3% BW in two equal meals. The endogenous AA losses were the mean results of three previously experiments determined by a same nitrogen-free diet fed to pigs. Pigs had free access to water during the experiment.ResultsThe KE supplementation improved (p<0.05) the AID and SID of Met, Thr, Val, Asp, Cys, and Tyr in rice bran. Inclusion of KE increased (p<0.05) the AID and SID of Met and Val in CSM. The KE supplementation decreased (p<0.05) the AID and SID of His in RSM and all measured AA except for Arg, Met, Trp, Val, Gly, and Pro in corn DDGS. There was an increase (p<0.05) in AID and SID of Leu, Ile, Met, Ala, Cys, Ser, and Tyr in PNM supplemented with KE compared with that without KE. Inclusion of KE increased (p<0.05) the AID and SID of crude protein, Leu, Ile, Phe, Thr, Asp, and Ser in cSBM.ConclusionThis study indicated that KE had different effects on ileal AA digestibility of feedstuffs for growing pigs, which can give some usage directions of KE in swine feed containing those detected feedstuffs.

Efficacy of crude and immobilizedenzymes from Bacillus licheniformis for production of biodegraded feather meal and their assessment on chickens

Abstract Keratinase enzymes are a special type of protease that has a bio-degradative potential for degrading keratin-containing substrates by the enzymes they produced during bioprocessing. The study was carried out to investigate the effect of microbial degraded feather meal on broiler chickens. The strain was previously isolated from a feather dumping site. The effects of crude and immobilizedenzyme-degraded feather meal were investigated on growth performance, haematology and intestinal histology of broiler chickens. Maximum activity of the keratinolytic enzyme was at 45 °C, the maximum bio-degradative potential at 48 h of fermentation, while the pH 7 exhibited the maximum keratinolytic activity. The values obtained for feed conversion ratio for birds on feather meal were statistically similar to the value obtained for those on the control diet. The microbial biodegradation of feather wastes could be a better approach to overcome high feed cost and environmental pollution arising from solid waste disposal.

Studies on potential application of crude keratinase enzyme from Stenotrophomonas sp. for dehairing in leather processing industry

Studies on potential application of crude keratinase enzyme from Stenotrophomonas sp. for dehairing in leather processing industry

Microbial Keratinase Production and Application to Improve the Properties of Wool Fabrics

Keratinases have been used because of their ability to separate keratin that cannot be broken down by common proteases in the processing of wool fabrics in the textile industry. In this study, to enhance the keratinase production from Streptomyces sp. 2M21, three different detergents (SDS, Triton X-100, Tween 80) in two different concentrations (0.5%, 0.1%) were added to the fermentation medium containing raw sheep wool as a solid substrate and results were evaluated compared to the control groups. The highest enzyme activity was obtained from 0.5% Tween 80 containing fermentation medium that increased enzyme activity from 111 (U/mL) to 399 (U/mL). Subsequently, the produced keratinase and commercial enzyme were compared for improving the hydrophilicity, dyeing and shrinkage properties of wool fabrics. Results suggest that, the keratinase produced from raw wool is feasible for processing of wool fabrics in textile industry.

Bio-prospecting of marine-derived fungi with special reference to production of keratinase enzyme - A need-based optimization study

Bio-prospecting of marine-derived fungi with special reference to production of keratinase enzyme - A need-based optimization study

Separating Keratinase Producer Bacteria from the Soil of Poultry Farms and Optimization of the Conditions for Maximum Enzyme Production

Feather is considered one of the environmental pollutant factors that can be hydrolyzed by bacteria and fungi. About 90% of the full weight of a feather consists of keratin. The structure of feather is very difficult to break down. Some bacteria in the presence of keratin-contained substrates are able to produce the keratinase enzyme to hydrolyze keratin. A total of 15 soil samples were collected from the poultry farms around Marvdasht city. 7 bacterial strains were grown in feather filled environment. 5 isolates that showed a clear analysis were selected and identified using biochemical tests and molecular methods. The bacterial DNA contents were sequenced and were assigned certain numbers in GenBank as new strains. Then, every fifth bacteria were also evaluated for the production of keratinase. All strains belonged to different strains of Bacillus. Five strains were able to completely degrade feather. Bacillus cereus SKH1 had the maximum enzyme activity, 17.12 (Unit/ml/min). Different strains of Bacillus in this study showed the ability to produce keratinase in the presence of keratin. Keratinase measurement showed that all 5 strains are potentially able to treat feather-contained waste.

Versatility and commercial status of microbial keratinases: a review

The world’s increasing population and shortage of food and feed is creating an urgently for us to look for new protein sources from waste products like keratinous waste. Poor management of these wastes has made them one of the major recalcitrant pollutants in nature. Microbial keratinases offers an economic and eco-friendly alternative for degrading and recycling keratinous waste into valuable byproducts. Diverse groups of microorganisms viz., bacteria, fungi and actinomycetes have the ability to degrade recalcitrant keratin by producing keratinase enzyme. Microbial keratinases exhibits great diversity in its biochemical properties with respect to activity and stability in various pH and temperature ranges as well as in the range of recalcitrant proteins it degrades like those present in feathers, hairs, nails, hooves etc. Owing to diverse properties and multifarious biotechnological implications, keratinases can be considered as promising biocatalysts for preparation of animal nutrients, protein supplements, leather processing, fiber modification, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical, cosmetic and biomedical industries, and waste management. This review article presents an overview of keratin structure and composition, mechanism of microbial keratinolysis, diversity of keratinolytic microorganisms, and their potential applications in various fields.

Production, partial optimization and characterization of keratinase enzyme by Arthrobacter sp. NFH5 isolated from soil samples

The study was conducted to select the best promising keratinolytic bacterial strain. A good keratinase positive bacterium isolated from the soil samples of Hazaribagh tannery industrial zone, Dhaka was identified as Arthrobacter genus depending on the conventional techniques and confirmed as Arthrobacter sp. by sequencing 16S rRNA gene. The medium components and culture conditions were optimized to enhance keratinase production through shake flask culture. Keratin and feather powder (10 g/l or 1%) were good substrates for the highest keratinase production along with yeast extract (0.2 g/l or 0.02%) as an organic nitrogen source and potassium nitrate (1 g or 0.1%) as an inorganic nitrogen source. Maximum yield of keratinase was found after 24 h of incubation at 37 °C with an initial pH of 7.0 and inoculums volume 5% under 150 rpm when keratin, yeast extract and potassium nitrate were used as nutrient sources. Keratinase production was more than 5.0-fold increased when all optimized parameters were applied simultaneously. The optimum reaction temperature and pH were determined to be 40 °C and 8.0 respectively for crude keratinase activity. Therefore, Arthrobacter sp. NFH5 might be used for large scale production of keratinase for industrial purposes in less time.

Dehairing of cattle hide by keratinase enzyme of Aspergillus flavus s125

Dehairing of cattle hide by keratinase enzyme of Aspergillus flavus s125

Evaluation of Keratinolytic Activity Succeeds by Keratinophilic Fungi in Jaipur, India

Earth has innate background for fungi that cover individual kingdom since evolution. The keratinophilic fungi are allied moulds that produce the keratinase enzyme to degrade the keratinous materials in or on the soil. Keratinous materials are insoluble and resistant to degradation by common proteinase enzymes. It is important to study the microorganism producers of such enzymes for use in the biotechnology industry. In order to present study, two isolates of fungi were evaluated to determine if they had the ability to degrade keratin as nutrient substrate. They were grown in an inundated culture medium containing poultry feathers. Among species, best keratin substrate degradation activity as well as keratinase enzyme activity was recorded in Arthoderma multifidium (KU578107) followed by Chrysosporium tropicum (KU578108) gradually leading manner day by days.

Preparation And Characterization Of Polyvinyl Alcohol–pectin Cryogels Containing Enrofloxacin And Keratinase As Potential Transdermal Delivery Device

Fil: Gonzalez, Jimena Soledad. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnologia de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingenieria. Instituto de Investigaciones en Ciencia y Tecnologia de Materiales; Argentina

Sulfitolytic and keratinolytic potential of Chryseobacterium sp. RBT revealed hydrolysis of melanin containing feathers.

In black feathers, melanin is embedded in keratin matrix that makes feather more resistance to the microbial degradation. Chryseobacterium sp. RBT previously isolated from the poultry waste disposable site revealed strong sulfitolytic and keratinolytic activities. Maximum keratinase activity was observed at 48 h (89.12 U ml−1) showed 83 % of native black feather degradation. The concentration of free sulfhydryl groups released during degradation was 0.648 × 10−4 M (12 h), 2.144 × 10−4 M (96 h), and however, declined on prolong incubation to 1.752 × 10−4 M (120 h). Melanin was released in the degradation medium after microbial exploitation of black feather. After purification, melanin was dark brown colored powder insoluble in water, 5 M HCL, ethanol, methanol, benzene, chloroform, and acetone; whereas, soluble in KOH and NaOH. On exposure to oxidizing and reducing reagents feather melanin showed decolorization, while formed a brown precipitate when reacted with FeCl3. The spectroscopic characterization of isolated melanin demonstrated absorption at infra-red region. Similarly, UV–visible scan confirmed that increase in the wavelength progressively declined the absorbance of pigment. The crude keratinase enzyme (2 % v/v) produced during degradation showed complete dehairing of goat skin within 20 h.

Cloning, expression and structure simulation of keratinase from Bacillus licheniformis strain MZK05

Cloning, expression and structure simulation of keratinase from Bacillus licheniformis strain MZK05

Anti-fungal potential of ozone against some dermatophytes.

Dermatophytes are classified in three genera, Epidermophyton, Microsporum and Trichophyton. They have the capacity to invade keratinized tissue to produce a cutaneous infection known as dermatophytoses. This investigation was performed to study the effect of gaseous ozone and ozonized oil on three specific properties of six different dermatophytes. These properties included sporulation, mycelia leakage of sugar and nutrients and the activity of their hydrolytic enzymes. Generally, ozonized oil was found to be more efficacious than gaseous ozone. Microsporum gypseum and Microsporum canis were the most susceptible, while Trichophyton interdigitale and T. mentagrophytes were relatively resistant. The study revealed a steady decline in spore production of M. gypseum and M. canis on application of ozonated oil. An increase in leakage of electrolytes and sugar was noticed after treatment with ozonized oil in the case of M. gypseum, M. canis, T. interdigitale, T. mentagrophytes and T. rubrum. The results also revealed loss in urease, amylase, alkaline phosphatase, lipase and keratinase enzyme producing capacity of the investigated fungi.

Isolation and identification of feather degrading bacteria from feather-dumped soil

Feathers contain 90% of protein in the form of keratin. Feathers are generated in large quantity as a by-product in poultry farms leading to environmental pollution. As chemical treatment process is expensive and does not completely degrade the feather wastes, an alternative process would be required. Microbial process is economical compared to chemical process, and it also helps convert feathers into value added products such as nitrogen fertilisers, feedstuffs, films and rare amino acids. The soil samples for the wastes were collected from a poultry dump area in Trichy. They were serially diluted and 50 microorganisms were obtained. The microorganisms obtained from the feather-dumped soil were inoculated on solid agar plates at 30°C for 24 h. Out of those, 10 organisms showed a clear zone of hydrolysis. The collected samples of soil were tested and screened to isolate pure keratinolytic strains. Clear zone of hydrolysis in milk agar medium confirmed the presence of proteolytic activity of organisms. The positive strain 1 which produced keratinase enzyme was identified as Bacillus cereus by 16S rRNA analysis.

Novel Keratinolytic Activity of Cyberlindnera fabianii Nrc3 Aza as A Plant Growth Promoting Agent (PGPA)

Two field experiments had been conducted in Nubaria sandy soil, Behaira Governate, Egypt to show the effect of keratinase enzyme produced by the novel microbial isolate (Cyberlindnera fabianii NRC3 Aza) on plants.The trials had been conducted in the two successive summer seasons (2011/2012 and 2012/2013) to show the effect of keratinase enzyme from degraded feather–waste on the morphology and chemical composition of peas pods (Pisumsativum L.)–family Fabaceae (Leguminasae). In 2011/2012 season, only the chemical analysis of the dried powdered beads was studied. In 2012/2013 season, the morphological studies of the yield were considered beside the chemical ones. The results depicted significant effects of the sprayed enzyme (keratinase) on peas as plant growth promoting agent (PGPA), compared with the blank (sprayed with water). Electrophoreses and amino acid analysis were carried out for the characterization of the partial pure keratinase enzyme. Int J Appl Sci Biotechnol, Vol 3(4): 609-618