Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent

Background A wide variety of bacterial species produces protease enzyme, and the application of the same enzyme has been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen, and identify alkaline protease-producing bacteria that were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia. Purpose To isolate and characterize the alkaline protease-producing bacteria from leather industrial effluents. Methods Samples are collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolated protease-producing bacteria using skim milk agar media. After studying primary and secondary screening using zonal inhibition methods to select potential protease-producing bacteria using skim milk agar media. Finally, to identify the potential bacteria using biochemical methods, bacterial biomass, protease activity, and gene sequencing (16S rRNA) method to finalize the best alkaline protease producing bacteria identified. Results First twenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at the Modjo town of Ethiopia. The isolated bacteria were screened using the primary and secondary screening method with skim milk agar medium. At the primary level, we selected three isolates namely ML5(14 mm), ML12(18 mm), and MS12 (15 mm), showing the highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to primary screening. Further secondary screening confirmed that the zone of inhibition methods ML5 (14.00±0.75 mm), ML12 (19.50±0.66 mm), and MS12 (15.00±1.32 mm) has efficient proteolytic activity and can be considered as effective protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species, and all the three bacterial isolates were found out to be of Bacillus species. The shake flask method was carried out to identify the most potent one having greater biomass production capabilities and protease activity. ML12 isolated from leather effluent waste showed the highest protease activity (19 U/ml), high biomass production, and the same was subjected to molecular identification using 16s sequencing and a phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 (Bacillus cereus strain -MN629232.1) is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1). Conclusions This study has exposed that from twenty-eight different bacterial samples isolated from leather industry effluent; further primary and secondary screening methods were selected three potential alkaline protease strains. Finally, based on its biochemical identification, biomass, and protease activity, ML12 (Bacillus cereus strains) is the best strain identified. The alkaline protease has the significant feature of housing potent bacterial species for producing protease of commercial value.

The Characterization of Stenotrophomonas maltophilia Isolated from Marine Sponge Producing Salt Tolerant Proteases

Protease is an important industrial enzyme and salt tolerant protease in which has desirable properties that could enhance its uses not only in industries, but also in agriculture and environmental. Marine organisms usually harbour halophilic microorganisms which produce salt tolerant protease. In this study, salt tolerant protease producing bacteria from marine sponge were isolated and screened on skim milk marine agar supplemented with different NaCl concentrations (1.5 % w/v). Out of 11 isolates, 8 isolates (S1-1, S1-2, S1-3, S2-1, S2-3, S2-4, S2-5 and S2-6) showed clearing zones with ability to digest casein on the skim milk agar. Morphologically, these strains are gram negative bacilli which grow in yellow colonies and were found to be catalase-positive but oxidase-negative. They are also non-lactose fermenter that produce gelatinase but not α-amylase. The ribosomal 16S rRNA sequencing was used to identify each isolate (Acc. number of S1-1 for MT645770, S1-2 for MT645 771, S1-3 for MT645 772, S2-1 MT645 773, S2-3 MT645 774, S2-4 MT645 775, S2-5 MT645 776 and S2-6 MT645 7767). The 16S rRNA sequences showed that these isolates were highly similar to Stenotrophomonas maltophilia (S2-6, 99.87%) and to a related strain Pseudomonas hibiscicola (S1-1, S1-2, S1-3, S2-1, S2-3, S2-4, S2-5 (97.16-99.9 %). Further proteolytic studies were carried out using skim milk agar with 1.0 %, 2.0 %, 3.0 % and 4.o % (w/v) of NaCl concentrations. All isolates were able to hydrolyze casein which produced clear zones surrounding each colony at 1.0 % and 2.0 % (w/v) salt. However, only isolate S1-5 and S2-6 showed proteolytic activities at 3.0 % (w/v) salt but none of them at 4.0 % (w/v). The ability of these isolates to produce protease which active at higher salt may indicate their potential to be the sources for enzyme with useful properties.

Isolation, Screening, Characterization and Identification of Alkaline Protease Producing Bacteria from Leather Industry Effluent

BackgroundA wide variety of Bacterial species produces protease enzyme and the application of same enzyme have been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen and identify protease producing bacteria which were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia.PurposeTo isolated alkaline protease producing bacteria from leather industrial effluents and to characterization (Secreening and identification).MethodsSample collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolate protease producing bacteria using skim milk agar media. After studying Primary and secondary screening using zonal inhibition methods to select potential protease producing bacteria using skim milk agar media. Finally to characterization and identification of potential bacteria using biochemical methods, protein estimation, biomass, protease assay and gene sequencing (16S rRNA) method to finalized best protease producing bacteria. ResultsTwenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at Modjo town of Ethiopia. The isolated bacteria were screened using primary screening method with skim milk agar medium. Three isolates namely MS12, ML5 and ML12 showing highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to secondary screening. Further secondary secreening confirmed that MS12, ML5 and ML12 has efficient proteolytic activity and can be considered as potent protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species and all the three bacterial isolates were found out to be of Bacillus species. Shake flask method was carried out to identify the most potent one having greater biomass production capabilities, protein quantity and protease activity. ML12 isolated from leather effluent waste showed highest Protein(170mg/ml), Protease activity(19U/ml), high biomass production and the same was subjected to molecular identification using 16s sequencing and a Phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1).ConclusionsThis study has revealed that the leather industry effluent site has significant feature of housing potent bacterial species producing protease of commercial value. Being one among the most widely used enzyme, comparatively. Protease holds a larger scope for research and commercialization any other type of enzymes. There is a need to develop novel protease enzymes for further necessary applications of these enzymes. Moreover, enzyme produced by bacteria which are present in effluents are a greater boon to establish the significance of converting industrial wastes to a highly valuable enzymes especially like proteases.

ISOLASI DAN KARAKTERISASI RHIZOBAKTERI YANG BERPOTENSI SEBAGAI AGEN PEMACU PERTUMBUHAN TANAMAN

Penelitian bertujuan untuk mendapatkan isolat rizobakteri yang memiliki aktifitas melarutkan fosfat, memproduksi enzim protease, dan memiliki aktifitas anti-fungal terhadap cendawan fitopatogen Fusarium oxysporum secara in vitro. Isolasi rizobakteri dilakukan di Laboratorium Perlindungan Tanaman Fakultas Pertanian Universitas Borneo Tarakan dari sampel tanah yang diambil dari daerah di sekitar Kota Tarakan, Kalimantan Utara dengan media Nutrient Agar (NA). Isolat bakteri yang diperoleh diuji keamanannya sebagai agens hayati dengan cara menumbuhkan benih timun yang telah direndam dalam suspensi bakteri. Bakteri yang aman (tidak berpotensi sebagai fitopatogen) kemudian dikarakterisasi fenotipnya dan sifat fisiologisnya. Sifat fisiologis yang diamati adalah kemampuan dalam menghasilkan enzim protease (pada media Skim Milk Agar, Merck, Germany) dan kemampuannya dalam melarutkan fosfat (pada media Pikovskaya Agar, HiMedia, India). Selanjutnya juga diuji kemampuannya dalam menghambat pertumbuhan F. oxysporum secara in vitro. Hasil penelitian meunjukkan bahwa diperoleh 35 isolat rhizobakteri yang non patogenik, 23 isolat rhizobakteri yang memiliki aktivitas proteolitik, 2 isolat rhizobakteri yang mampu melarutkan Fosfat dan tidak ada isolat rhizobakteri yang memiliki kemampuan antifungal.

Enterococcus faecium 1.15 Isolated from Bakasam Showed Milk Clotting Activity

The Lactic Acid Bacteria with Milk Clotting Activity (MCA) were isolated from Bakasam, an Indonesian traditional fermented meat. The isolate screening was carried out using modified method of Skim Milk Agar and Milk Clotting Activity Test, and the isolate was then identified using 16S rRNA. We found 4 isolates that showed MCA of 18-20 SU/ml. Identification using 16S rRNA indicated that the isolate ALG.1.15 was 99% (FR3-F primer) and 99% (FR3-R primer) identic with Enterococcus faecium. The isolate potentially produced renin-like protease to subtitute renin from veal.

PRODUCTION OF KERATINASES FROM NOCARDIOPSIS SP. 28ROR AS A NOVEL IRAQI STRAIN

Objectives: isolate a novel feather- degrading actinobacterial species had the ability to produce wide pH activity keratinases.Methods: Of 23 actinobacterial isolates were recovered from farm soil, poultry farm soil and feather wastes, these isolates were screened for protease and keratinase production on skim milk agar, feather media, and antimicrobial production. One potential isolate was identified depending on phenotypical, physiological and molecular according to partial sequences of 16S r RNA gene analysis and optimized keratinase production. Results: 11 isolates out of 22 protease producer had the ability to degrade raw chicken feather and some of these isolates produced antifungal and antibacterial metabolites.The potential isolate, Nocardiopsis sp. 28ROR (GenBank: KC702802.1), produced two types of extracellular keratinases in feather meal medium at pH6 (acid type), 30-35°C for 7d and the alkaline keratinase at pH10, 40°C for 7d.Conclusion: The Nocardiopsis sp. 28ROR was a novel strain produced keratinases using feather meal degradation as a cheap waste medium. The wide tolerance of temperature and pH by keratinase makes it an ideal contender to be investigated further for potential application as a detergent additive.Keywords: Nocardiopsis, Keratinase, Optimization, Feather medium, Antibiotic.

ISOLASI BAKTERI PENGHASIL PROTEASE ASAL KAWASAN TAMAN HUTAN RAYA SULTAN SYARIF HASYIM RIAU

Taman Hutan Raya Sultan Syarif Hasyim, Riau (Tahura SSH) merupakan salah satu hutan wisata dengan beranekaragam organisme yang memiliki potensi sumber daya alam. Rangkaian kegiatan pemanfaatan sumber daya alam meliputi kegiatan eksplorasi potensi mikroorganisme penghasil enzim protease. Protease merupakan salah satu enzim yang paling banyak digunakan dalam industri. Isolasi bakteri penghasil protease dilakukan dengan menggunakan medium Skim Milk Agar (SMA) 0,5%. Pengujian isolat didasarkan pada aktivitas zona bening secara semikuantitatif berdasarkan uji nilai tengah. Hasil penelitian diperoleh 12 isolat bakteri yang mampu menghasilkan enzim protease. Berdasarkan uji nilai tengah diperoleh 3 isolat kriteria tinggi penghasil protease yaitu isolat St₁P₈ sebesar 55,7 mm, isolat St₁P₄ sebesar 53,7 mm, dan isolat St₁P₂ sebesar 47,7 mm.

Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore

In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

Proteolytic and Lipolytic Activity of Molds Isolated from Aged Beef

This study evaluated the proteolytic and lipolytic activity of several strains of Thamnidium elegans, Mucor mucedo and Chaetostylum fresenii on selected test proteins and lipids. At 18°C, the zone of hydrolysis to colony size ratio on skim milk agar, representing proteolytic activity after 4 d, was 0.92, 0.80 and 0.67 for M. mucedo, C. fresenii and T. elegans, respectively. A similar trend was noted after 4 d of incubation at 24°C. There was positive lipolytic activity on Tween 80 at 18 and 24°C for the same three molds. The proteolytic and lipolytic activity decreased with decreasing temperatures so that at 4°C, the temperature of most probable use if applied to meat, the effect was negligible unless long incubation times were used. The absence of proteolytic activity of the molds at 4°C and the impracticality of aging beef at 18 or 24°C suggest that treatment of meat with molds to enhance tenderness may not be feasible.