Milk Agar Medium Research Articles

Bio-production of alkaline protease by Trichoderma longibrachiatum and Penicillium rubidurum using different agro-industrial products

Alkaline protease being active in neutral to alkaline pH has huge demands in food, detergent, leather and pharmaceutical industries. Its production from agro-industrial wastes not only lowers the production costs but also reduces the environmental problems. Hence, the present study aimed to search for new potential microbes, which can produce alkaline protease enzyme, to meet the industrial demands. In this study, 13 fungal spp. were isolated on potato dextrose agar medium (PDA) from mangrove soil through serial dilution, and then were streaked on the skim milk agar medium for qualitative screening of protease production. Out of 13 fungal spp.; only 7 spp. were able to produce proteolytic zones through the proteolytic assay. The Relative enzymatic index (REI) value (Zone diameter/Colony diameter) of all the fungal isolates that produced proteolytic zones on skim milk agar medium was evaluated. Only 2 fungal isolates which showed maximum REI value were selected, and then identified morphologically and molecularly as Trichoderma longibrachiatum (Accession no. MF144551) and by Penicillium rubidurum (Accession no. MF144561). Submerged fermentation was carried out using different agro industrial substrates to quantify for protease production, where the supernatants obtained were used for alkaline protease estimation. Among the different tested substrates, soybean powder and wheat bran were the most suitable substrates for maximum protease production by T. longibrachiatum (233.78±7.12 U/ mg) and P. rubidurum (228.61±11.13 U/ mg), respectively. The partial purified enzyme from these fungi showed maximum proteolytic potentials at pH 8.0 (P. rubidurum) and pH 9.0 (T. longibrachiatum), with optima temperature of 40 °C. Among the tested heavy metals, only Mn2+ expressed marginal enhancement of the protease enzyme activity.

Proteolytic and Clot Lysis Activity Screening of Crude Proteases Extracted from Tissues and Bacterial Isolates of Holothuria Scabra

Cardiovascular diseases (CVDs) are still the leading mortality causes in the last decades. The ailments are multifactorial characterized by excessive clot (thrombus) formation in the blood vessels. Thrombus could be degraded through thrombolysis mechanism by plasmin activated by various fibrinolysis agents including, urokinase, nattokinase, or streptokinase. However, the use of these agents is restricted by relatively high cost, short half-life, allergic reaction, and bleeding effects. The search for more economical and safer thrombolytic (clot lysis) agents are essential to address the underlying problem in CVD therapy. Among Holothurians, H. scabra has been known to have the highest protein content making it ideal substrate for protease enzymes including fibrinolytic types with clot lysis properties. However, isolation of a proteases with antithrombotic activities either from tissue or from bacteria of H. scabra has not been reported. This study aimed to screen proteolytic and clot lysis activities of crude protease extracts from tissue and bacteria isolated from fermented intestine of H. scabra. Crude protease of tissue of H. scabra was extracted by cold centrifugation, which activity was measured using UV-spectrophotometer. Crude protease of proteolytic bacteria selected by clear zones on skim milk agar (SMA) medium was isolated from nutrient broth (NB). Extracts showing proteolytic activity were subjected to gravimetry-based clot lysis test. As results, crude proteases isolated directly from the H. scabra’s tissue showed low proteolytic activities, thus were no proceed to clot lysis activity test. Crude protease extracted bacteria could show both proteolytic and clot lysis activities. In conclusion, based on this screening study, intestine of H. scabra is a rich source of proteolytic bacteria, some of them could produce crude enzymes showing competitive thrombolysis activities with Nattokinase. Thus, they appeared to have more potentials to be developed as thrombolysis agent than those directly extracted from the organism’s tissue.

Isolation, characterization, activity test and molecular identification of thermophilic bacteria producing proteases from Dolok Tinggi Raja Natural Hot Springs, North Sumatra, Indonesia

Abstract. Fachrial E, Krisdianilo V, Harmileni, Lister INE, Nugroho TT, Saryono. 2021. Isolation, characterization, activity test and molecular identification of thermophilic bacteria producing proteases from Dolok Tinggi Raja Natural Hot Springs, North Sumatra, Indonesia. Biodiversitas 22: 1725-1732. Proteases are complex enzymes that can be applied in both commercial products and physiological fields. This enzyme accelerates the breakdown of peptide bonds in polypeptides and proteins using hydrolysis reactions, transforming them into simpler bonds. In recent years, the demand for protease enzymes in Indonesia is still dependent on imported products, and there has also been an increase in their use. This is because there are many advantages in terms of efficiency, environmental friendliness, and its ability to catalyze a reaction without by-products. Proteases dominate the enzyme market with a total of 70% sales. Furthermore, they are manufactured from thermophilic bacteria as a solution to the problem of enzyme use in industries, where enzymes are easily damaged at high temperatures. The thermophilic protease enzyme can be found in thermophilic microorganisms living in extreme temperatures such as hot springs. There are lots of mountainous areas in North Sumatra Province, Indonesia with many hot springs. For example, the Dolok Tinggi Raja Natural Hot Springs of Simalungun District which has a water temperature of 50oC with a pH of about 6, the potential of which still needs to be explored. This study aims to isolate and determine the activity and molecular structure of thermophilic bacteria producing protease in Dolok Tinggi Raja Natural Hot Springs. Protease activity was determined based on the formation of clear zones in Skim Milk Agar medium. In addition, a total of 20 isolates were successfully obtained from sediment and water. Each was tested biochemically and morphologically, where 19 isolates had the activity of proteases, with the highest in isolate UTMTR VK S9 of 23.67 U/mL. Molecular identification was performed by the amplification of 16SrRNA gene based on BLAST and phylogenic analysis with MEGA X. Phylogenic results showed that isolate UTMTR VK S9 possessed similarities to Bacillus paralicheniformis strain A30103. Meanwhile, more research is required to purify enzymes for application in the industrial world.

Effect of Acridine Orange on Pathogenicity of E. coli Isolated from Urinary Tract Infection Patients

A total of (108) isolate from Urinary tract infection (UTI) patients, only (47) isolate gave a positive result(43.51%) after a number of morphological and biochemical characterization. Our result showed only 28isolate have the ability for protease production and haemolysin production (59.5%), and E. coli no.6 wasthe highest protease production isolate (24mm of lysis area) while the E. coli no.18 was lowest proteaseproduction (12 mm of lysis area). After incubtion on skim milk agar medium at 37°C for 18h and with(100%) haemolysin activity for all 28 isolates after growing on blood agar medium. In our study, firstattempt made on the effect of acridine orange (0.1%) as a curing agent on virulence factors of pathogenicbacteria ( both protease and haemolysin productin) by highly producer E. coli no.6 at concentrations ( 0, 10-1to 10-10) and the results showed that E. coli no.6 loss the protease and haemolysin productin at concentrationof (10-1 to 10-4) while little and normal activities were observed at concentration of (10-5 to 10-10) of acridineorange. Also, this study was investigate the agarose - gel electrophoresis of both cured and normal cells andthe results showed the presence of both chromosomal and plasmid bands in the normal cells and only thepresence of chromosomal band for the cured cells E. coli treated with acridine orange at concentrations of(10-2 to 10-4).

KOSAKONIA SP. PROTEOLYTIC BACTERIA ISOLATED FROM RUMEN AND RETICULUM OF ACEH CATTLE

The aim of this study was to identify proteolytic bacteria from the ruminal and reticulum fluids of aceh cattle based on the 16S rRNA gene. Samples used were ruminal and reticulum fluids of aceh cattle slaughtered in Abattoir of Aceh Besar. Samples were diluted and cultured into Skim Milk Agar medium at 39 C for 48 hours. The morphology of bacterial colonies growth in the medium was observed. Colonies resulted in the largest clear zone were isolated and used for Deoxyribonucleic Acid (DNA) isolation, 16S rRNA gene amplification and sequencing. Theresults showed that morphology of dominant colonies was yellowish white color, round shape, position on the agar surface. The results ofphylogenetic analysis of RS1 and ReS2 isolates isolated from rumen and reticulum fluids of aceh cattle respectively had a close familialrelationship and belonged to the bacterial group of Kosakonia. Sequence homology showed isolate RS1 and ReS2 are probably either newEnterobacteriaceae species or unconfirmed species. Halo zone produced by ruminal bacteria had a wider diameter (25 mm vs 20 mm) than thatcaused by reticulum bacteria). Based on the results, RS1 (bacterium in the rumen) and ReS2 (bacterium in the reticulum) belong to similar type, namely Kosakonia sp. with a proteolytic activity. Presumably, these bacteria originate from the rumen that enters the reticulum with degraded feed.

Optimization of Alkaline Protease Production in Submerged Fermentation Using Bacillus cereus Isolated from an Abattoir Wastewater in Ile-Ife, Nigeria

This study isolated and screened protease-producing bacteria from abattoir wastewater, it determined the effect of physical and nutritional components on the production of the crude protease produced by a selected bacterium in order to obtain a bacterium capable of producing protease with properties for industrial applications. Abattoir wastewater samples were collected from different locations in Ile-Ife, Nigeria and using pour plate method, one milliliter each of the aliquot was inoculated into skimmed milk agar medium (SKMA) to obtain colonies of protease-producing bacteria after proper dilution. All the protease-producing bacterial isolates were further screened for protease activity by submerged fermentation technique. The growth curve and protease activity of the best isolate was determined. The effect of different components of the growth medium on protease production was also determined. Forty-three isolates were obtained, five of the isolates gave appreciable diameter of clear zones of hydrolysis by rapid plate assay technique, out of which Bacillus cereus gave the highest enzyme activity of 75.79 Units/ml/min by submerged fermentation process. The optimal production of the enzyme was obtained after 44 hours of incubation with an inoculum volume of 2.0 ml. The optimal pH and temperature of production were 10.0 and 50°C respectively, while the best carbon and nitrogen source was sucrose and NH4Cl respectively. The study concluded that abattoir wastewater served as a good media for the growth of proteolytic bacteria while the strain of Bacillus cereus isolated could be applied in the industries for protease production.

Isolation and molecular identification of carbohydrase and protease producing Bacillus subtilis JCM 1465 isolated from Penen Hot Springs in North Sumatra, Indonesia

Abstract. Fachrial E, Anggraini S, Harmileni, Nugroho TT, Saryono. 2019. Isolation and molecular identification of carbohydrase and protease producing Bacillus subtilis JCM 1465 isolated from Penen Hot Springs in North Sumatra, Indonesia. Biodiversitas 20: 3493-3498. The application of enzymes industrially is increasing every year and thermophilic microbes are a promising source of these enzymes for industrial use due to their temperature stability. The aim of this study, therefore, was to isolate, characterize and identify the enzyme producer-thermophilic bacteria from the Penen Hot Spring in Deli Serdang, North Sumatra, Indonesia. In the experimental setup, carbohydrase activities including inulinase and amylase were determined by the formation of clear zone around the colonies after soaking with Lugol on the TSA medium supplemented with 1% inulin powder and 1% starch. Similarly, the protease activities were determined by the formation of clear zone around the colonies on Skim Milk Agar Medium. Then, of the 11 isolates, only one known as UTMP 12 showed maximum enzyme activity. The isolate was then characterized based on morphology and biochemistry and found to be Bacillus subtilis strain JCM 1465 (accession number NR_113265, homolog 99.72%). Furthermore, this is the first study on carbohydrase and protease activities of Bacillus subtilis strain JCM 1465 and the result shows that the thermophilic bacteria are needed in the production of carbohydrase and protease.

Constraints in Adoption ofModern Farm Machines by Tribal Farmers in Ramgarh District of Jharkhand

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) andamp; the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method andamp; it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column andamp; the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , andamp; one normal cell line Ref ( Rat embryonic fibroblast ). The cancer andamp; normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8andamp; 0.16 mg/ml) then incubated for additional 48h at 37C 0 andamp; the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andamp;slight toxicity ( 37.12% )on normal cell line (Ref) in a concentration (0.8mg/ml).

Cytotoxic Effect of Pseudomonas aurogenosa Protease on Cancer Cells

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) and the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method and it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column and the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , and one normal cell line Ref ( Rat embryonic fibroblast ) . The cancer and normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8and 0.16 mg/ml) then incubated for additional 48h at 37C0 and the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andslight toxicity ( 37.12% ) on normal cell line (Ref) in a concentration (0.8mg/ml).

Screening and Characterisation of Keratin-Degrading Bacillus sp. from Feather Waste

Aim: This study focuses on the screening and characterisation of keratin-degrading Bacillus species from feather waste.
 Methods: Nine bacteria were isolated from feather waste obtained from a poultry layout at Egbeda local government secretariat, Ibadan, Nigeria. These bacteria were grown in basal medium with feather as primary source of carbon, nitrogen, sulfur and energy. Feather degrading bacteria were screened for both proteolytic activity and keratin degradation on skimmed milk agar and keratin azure medium respectively. They were also screened for their ability to degrade other keratin substrates such as hair and nail.
 Results: Three of the isolates with higher feather degradation levels also showed high proteolytic activity and release of azure dye. They were selected and identified phenotypically and genotypically using 16S rRNA sequencing as Bacillus licheniformis-K51, Bacillus subtilis-K50 and Bacillus sp.-K53. The bacteria were capable of degrading other keratin-containing substrates such as nail and hair. Bacillus subtilis-K50 and Bacillus licheniformis-K51 showed significant difference (P) in degradation among the three different keratin sources used yielding higher degradation with feather as keratin source with respective optical densities of 0.07 and 0.11 followed by hair and least in nails with optical densities of 0.05 and 0.07 respectively. Highest degradation of all the three keratin substrates was observed in Bacillus licheniformis-K51.
 Conclusion: The three isolated bacteria possess the ability to degrade keratin and utilize feather as keratin substrate. As a result, these can be considered as potential candidates for degradation and utilization of feather keratin.

Isolation and identification of proteolytic bacteria from pig sludge and protease activity determination

Proteolytic bacteria were found in many environment, some were found in soil or mud. This research was conducted to find proteolytic bacteria from pig sludge that is place for pig wallowing. Bacteria isolation was carried out by streak plate method on nutrient agar media. Qualitative test of proteolytic bacteria used skim milk agar medium. The results of isolation obtained 10 bacterias and 2 of them have potential as proteolytic bacteria. Identification of proteolitic bacteria used the 16s rRNA method, result showed that bacterias are Bacillus pseudomycoides and Staphylococcus sciuri. Protease isolation was carried out under 4°C and under room temperature during centrifugation as separation process. Protease activity from Bacillus pseudomycoides was at 4°C as much as 0.094 U/mL and was 0.082 U/mL at room temperature, while from Staphylococcus sciuri was at 4°C as much as 0.120 U/mL and was 0.098 U/mL at room temperature.

Isolation and Characterization of Protease Producing Bacteria from Soil, in Arba Minch University, Abaya Campus

Microbes are ubiquitous and live in familiar settings such as soil, water, food and animal intestines as well as in more extreme setting such as rocks, glaciers, hot springs and deep-sea vents. They are essential component of the biodiversity as they account for 50% of the living biomass of this planet. The aim of this work was isolation and screening of protease producing bacteria from soil collected from the study area. Isolation of protease producing bacteria was performed by the serial dilution and spread plate technique using skim milk agar medium. Accordingly, 131 isolates were positive on skim milk agar and of which 13 (10%) of them are protease producer. Four best isolates were selected on the basis of their clear zone size, one from each site for further study. IS-4 has larger clear zone size with 10.5mm followed by IS-2 (6mm). All potential isolates have circular in shape of colony and flat in height of colony except IS-1 which is irregular and raised respectively. Most of potential isolates have opaque in their colonial optical density. All isolates were found to be Gram positive and spore producers. Morphological and biochemical results have revealed that three of four active enzyme producing bacteria are identified as Bacillus spp. Members of the genus Bacillus produce a large variety of extracellular enzymes (proteases) particularly significant for industrial application.

Production and Characterization of Extracellular Protease from Bacillus sp. 140-B Isolated from Pineapple Plantation in Lampung, Indonesia

<p class="Els-Abstract-text"><em>Bacillus </em>sp<em>.</em> being industrially important organisms produces a wide variety of extracellular enzymes including protease. <em>Bacillus </em>sp. 140-B isolated from rhizosphere area of pineapple plantation in Lampung Province, Indonesia was tested for production of protease on skim milk agar medium. The aims of this study were to characterize and investigate some properties of protease activity from <em>Bacillus </em>sp<em>. </em>140-B. <em>Bacillus </em>sp<em>. </em>140-B showed protease activity qualitatively by clear zone diameter of 15.0 mm and the highest protease activity was 35.02 Unit · mg<strong><sup>–1</sup></strong> protein at 14 h after incubation. Some properties of protease activities from <em>Bacillus </em>sp<em>. </em>140-B including effects of temperature, pH and several metals were observed in this experiment. The protease activity from <em>Bacillus </em>sp<em>. </em>140-B had optimum pH of 7,0 and the optimum temperature was 60 °C. Several metals which were evaluated on protease activity showed that Mn could increase protease activity, while other metals (Ca, Co, and Hg) showed decreasing protease activity of <em>Bacillus </em>sp<em>. </em>140-B.</p><div><p class="Els-keywords"><em> </em></p><p class="Els-keywords"><strong>Keywords: </strong><em>Bacillus sp.</em> 140-B; enzyme activity; enzyme properties; protease</p></div>

Isolation of Protease Producing Bacteria from Soil for Polyester and Silver Recovery from Waste X-ray Film

Proteases are multipurpose group of enzymes which mainly produced from microorganism and used in various industries such as detergent, silver recovery, food, pharmaceutical, leather and textile industries. This work aimed to isolate protease producing bacteria from local environment for use as silver recovery and reuse of polyester from waste X-ray film. Isolation of protease producer was undertaken using skim milk agar medium. A total of 36 protease positive bacteria were isolated from the study area (Arba Minch fish processing site). Five isolates were screened which had relatively higher clear zone and further examined gelatin removal efficiency of their crude enzymes. One isolate designated as <i>Bacillus sp.</i> THZ14 was selected on the basis of gelatin removal efficiency. The crude enzyme from this strain removed gelatin on waste X-ray film at pH 9, 40°C in 40 min. These properties suggest that protease from <i>Bacillus sp.</i> THZ14 could find potential application in silver recovery process and reuse of polyester sheet which have an economic implication.

Isolation and Screening of Protease Producing Bacteria from Local Environment for Detergent Additive

Proteases are among the most important hydrolytic enzymes that found in every organism to undertake important physiological functions. They are multipurpose enzymes used in various industries such as detergent, silver recovery, food, pharmaceutical, leather, and textile industries. This work aimed to produce protease from indigenous microbes for use as detergent additive. Isolation of protease producer was undertaken using skim milk agar medium. Crude enzyme was characterized in terms of wash and stain removal tests. A total of 188 protease positive bacteria were isolated from seven samples collected from Arba Minch town. Out of 36 alkaline protease producing bacteria, one isolate designated as Bacillus sp. Cab44 was selected. The optimum activity was reached at pH 9 and 50°C. The enzyme was stable in the pH range of 7 to 10. It retained 75%, 86% and 72% activity after one hr pre-incubation at 50°C, in 15% H2O2 and 0.3% commercial detergent respectively. The enzyme activity was increased by Mg2+, Cu2+, and Mn2+, was not affected by Ca2+ but decreased by Zn2+, Hg2+ and Fe2+. It removed stains of blood and egg yolk on cotton cloth at pH 9, 40°C, 5.07 U/ml in 30-40 min. These properties suggest that protease from Bacillus sp. Cab44 could find potential application in detergent industries as good candidate of additive in detergent formulation which have economic implication.

Utilization of Dairy Effluent for Food Grade Protease Production Using Bacillus &amp;lt;i&amp;gt;sp&amp;lt;/i&amp;gt;.

Food grade proteases are proteyolytic enzymes having application in baking, food processing, protein modification etc. As a commodity product, pressure on protease market is on prize reduction and increasing performance. Hence our objective was to isolate a potent protease-producing microorganism and formulate a cost effective medium for neutral protease synthesis by the potent microbial culture. In order to achieve the objective, a proteolytic bacterium was isolated from soil using milk agar medium and the bacteria was identified as <i>Bacillus</i> sp. by morphological and biochemical characterization. Dairy industry effluent was then studied as a medium for neutral protease synthesis by the potent bacteria. Supplementation of mineral salt to the medium did not show profound influence of environmental factors such as medium pH, incubation temperature, agitation rate and incubation time on enzyme production. Optimum enzyme titers were found at pH7 when incubated at 37°C and 120 rpm 48 h. Dairy industry effluent was thus found to be a cost effective medium for neutral protease synthesis by <i>Bacillus</i> sp.

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.

Isolation and Screening of Protease Enzyme Producing Bacteria from Cheese at Dilla University, Ethiopia

Enzymes are biological catalysts that facilitate the conversion of substrates into products by providing favorable conditions that lower the activation energy of the reaction. An enzyme may be a protein or a glycoprotein and consists of at least one polypeptide moiety. Proteases are the most important industrial enzymes and comprise about 25% of commercial enzymes in the world. Two third of the industrially produced proteases are from microbial sources. The current study was focused on the screening of protease enzyme producing bacteria from dairy product of cheese at Dilla University. Cheese sample was collected from Dilla town market. The proteases enzyme production bacteria were screened on milk agar plate through spread plate method. As the result, certain bacteria were screened for protease enzyme production as well as were confirmed on milk agar medium. Almost all isolated (35 isolate) of bacteria from cheese food stuff were had a potential in the production of protease enzyme which help for various activities. Therefore, all isolate had a promising potential for production proteolytic enzyme which are used in industries and the other sectors.

Isolation and Selection of Feather-Degrading Aerobic Bacteria From Poultry Processing Plants in Mekong Delta of Vietnam

Studies on keratinolytic microorganism have been mainly related to biotechnological applications based on their capability for decomposition of keratin-containing wastes to produce animal feed and bio-fertilizer. However, these organisms have ecological relevance to recycling keratinous residues in nature. This study aimed to select and identify new local potential feather - degrading bacteria for applications in biotechnological processes in Mekong Delta. A total of 21 aerobic, rod-shaped isolates from Mekong Delta poultry processing plants showed capability to growth on feather meal agar medium. They all expressed clear zones around the colonies on milk agar medium indicating proteolytic activity. They also hydrolyzed 24.43% to 58.13% of chicken feather dry weight after 7 days incubation at 37 o C and partially disintegrated the whole feather after 10 days. The isolates K15 and K14 showed the most prominent feather-degrading activity were further identified according to their morphology and physiological characteristics. Molecular identification based on their 16S rDNA gene sequence also carried out and revealed that K14 as Chryseobacterium indologenes ; whereas K15 was highly related to Bacillus subtilis.

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosine agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate (LK-4) was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as “Streptomyces espinosus strain LK4 (KF806735)”. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinase enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.