Production of β-Mannanase by Penicillium italicum Subjected to Different Growth Conditions

Aims: The process parameters affecting enzyme production were optimized to ascertain the best optimal conditions for β-mannanase production by Penicillium italicum in solid state fermentation. Study Design: Four stages of experimental processes were designed for this study. The first experiment, samples were withdrawn after 24, 48, 72, 96, 120, 144,168 and 192 h incubation. In second experiment, the fermentation media were incubated at different temperatures. In third experiment, the effect of different pH values on β-mannanase production was evaluated, while the fourth experiment described the supplementation of surfactants in mineral salt solution for β-mannanase production. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: β-mannanase production was conducted using Locust Bean Gum (LBG) as the sole carbon source; moisten with mineral salt solution, and enzyme activity determined by dinitrosalicylic acid method, while protein content was determined by Lowry method. Results: Maximum enzyme activity (146.389 U/ml) was observed after 72 h of incubation. Different surfactants were supplemented in the basal medium, and Sodium Dodecyl Sulfate (SDS) was observed to give the highest β-mannanase activity of 53.335 U/ml. Initial pH of the culture medium was optimized and a pH of 6.0 was found to support maximum enzyme activity (173.241 U/mg protein). The optimum incubation temperature was achieved at 35°C. Conclusion: The results obtained provide information on optimal process parameters that might improve the yield of β-mannanase by P. italicum for better fish feed formulation, especially in the larval stages of fish fingerlings when the enzyme system is not efficient.

Screening of Bacterial Strains of Agricultural Waste Origin for Βeta-mannanase Production and Optimization of Process Parameters.

Aims: This study was carried out to screen bacterial strains of agricultural wastes origin for β-mannanase production and optimization of culture conditions. Study Design: The first experiment, bacterial strains were screened for β-mannanase production. In the second experiment, the best incubation time was determined. In the third experiment, different agricultural wastes were screened. In the fourth experiment, different nitrogen sources were screened. In the fifth and sixth experiments described the effect of different pH values and incubation temperatures on β-mannanase production. The best moisture content was determined in the seventh experiment, while in experiment eight; effect of different inoculum concentrations was evaluated. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure, Nigeria between September 2011 and March 2012. Methodology: Bacterial strains were screened and β-mannanase production from optimization studies was conducted on Locust Bean Gum. Enzyme activity was determined by dinitrosalicylic acid method. Results: Out of the sixteen bacterial strains screened, Klebsiella edwardsii designated 1A was selected as the most potent in producing enzyme of high activity and it was therefore selected for further studies. Pineapple peels were found to be the most effective carbon source with a highest β-mannanase activity of 8.533±0.08U/ml. Ammonium nitrate (NH4NO3) was obtained to be the best nitrogen source out of all the nitrogen sources screened. The best moisture content was obtained at 1:11 (ratio of substrate to salt solution). Inoculum concentration of 1.0% (v/v) yielded highest β-mannanase activity of 15.833±0.01U/ml. Addition of simple carbon sources to medium containing LBG caused a catabolic repression of β-mannanase synthesis. Conclusion: The optimal culture conditions obtained from this study will help to standardize the requirements for optimum β-mannanase production using cheaper substrates.

Kinetic Properties of Purified β-Mannanase from Penicillium italicum.

Aim: The study aimed at purification and characterization of β-mannanase from Penicillium italicum. Study Design: The first experiment, β-mannanase from Penicillium italicum was produced in basal medium supplemented with Locust Bean Gum (LBG). The second described the purification of crude β-mannanase, while the third experiment dealt with characterization and kinetic studies of purified β-mannanase from Penicillium italicum. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between July and August 2012. Methodology: β-mannanase from Penicillium italicum was produced in basal medium supplemented with LBG. The enzyme was purified by ammonium sulphate precipitation, ion exchange chromatography (DEAE-Sephadex A-50) and gel filtration (Sephadex G-150). The purified enzyme was characterized to determine its optimal conditions by standard assay procedures. The kinetic parameters of the purified enzyme were determined by Lineweaver-Bulk plot. Results: Fractionation of ammonium sulphate precipitated β-mannanase from Penicillium italicum on sephadex A-50 produced one major activity peak. Further fractionation of partially purified enzyme from ion exchange on Sephadex G-150 yielded one activity peak. A pH of 5.0 was optimum for purified enzyme activity and relatively stable between 40 to 100 min of incubation at this pH. The optimum temperature was 70ºC and 100% thermostable for 40 min after which a slight decline in activity was observed. The apparent Km for the hydrolysis of LBG from Lineweaver-Bulk plot was approximately 0.26 mg/mL, while the Vmax was 0.12 μmol/min/mL. The incubation of salts and organic compounds at 10 mM and 40 mM caused inhibition of enzyme activity. At 20 mM, enzyme activity was enhanced by FeSO4.7H2O, SDS and ZnSO4. 7H2O, while others caused inhibition of enzyme activity. The incubation of enzyme with CaCl2 and FeSO4.7H2O at 60 mM enhanced enzyme activity, while others caused inhibition. Conclusion: The result obtained from this study revealed that purified β-mannanase is active over a wide pH and temperature, and its stability implies that the enzyme will be useful during industrial processes where extreme conditions are required.

Isolation and Screening of Mannanase Producing Bacteria from Agricultural Wastes.

Aim: The work focused on the isolation and screening of mannanase-producing bacteria associated with selected agricultural wastes. Study Design: The first experiment, mannanase-producing bacteria were screened for mannanase production on Locust Bean Gum (LBG) agar medium and total bacterial count was determined. In the second experiment, the isolated bacteria were further screened for mannanase production in submerged state fermentation. Place and Duration of Study: Microbiology Research Laboratory Federal University of Technology, Akure and Postgraduate Research Laboratory, Obafemi Awolowo University Ile-Ife, Nigeria between September 2011 and March 2012. Methodology: The associated bacterial isolates were isolated on agar medium containing LBG and counted by standard microbiological methods. Quantitatively, mannanase production was conducted in mineral salt medium into which copra meal had been incorporated as the sole carbon source and enzyme activity was determined by dinitrosalicylic acid method. Results: The highest bacteria counts were recorded in compost from wood dust with 5.5×1011 cfu/g, while cassava peels had the least of 1.02×106 cfu/g. In this study, 23 bacterial isolates showed positive results with clear zone around the cultures. Bacterial isolate 1A showed the highest ratio of clear zone to colony, while the lowest was observed in isolate 4B. In liquid broth, all the 23 isolates displayed mannanase activity between 0.28 to13.89 U/ml for static and 0.56 to 13.43 U/ml for shaken condition, with the highest mannanase activity observed with isolate IA for both culture conditions. In the comparative study between static and shaken conditions, it was revealed that shaken cultures exhibited better yield than static cultures. According to the morphological and biochemical studies, the isolate 1A was primarily identified as the Klebsiella edwardsii. Conclusion: In this investigation, bacterial isolates evaluated for mannanase production from agricultural wastes elaborated considerable mannanase activity and this could be applied in feed and prebiotic.

Screening and Identification of Mannanase- Producing Fungi Isolated from Selected Agricultural Wastes.

Aim: The study evaluated potential performance of different fungal isolates from agricultural by-products for mannanase production. Study Design: The first experiment, fungal isolates were screened for mannanase production on agar medium containing Locust Bean Gum (LBG) and total fungal count was conducted. In the second experiment, the fungal isolates were further screened for mannanase production in submerged state fermentation. Place and Duration of Study: Microbiology Research Laboratory Federal University of Technology, Akure and Postgraduate Research Laboratory, Obafemi Awolowo University Ile-Ife, Nigeria between September 2011 and March 2012. Methodology: The fungal isolates associated with some agricultural wastes were isolated on LBG containing agar medium by plate assay techniques and counted by standard microbiological methods. Mannanase production was conducted in submerged state fermentation (shaken & static) into which copra meal had been supplemented as the sole carbon source and enzyme activity was determined by dinitrosalicylic acid method. Results: In this study, 11 fungal isolates showed positive results with clear zone around their cultures. Fungal isolate 5A showed the highest activity ratio of 1.8, while the least was observed in isolate 9A12 with activity ratio of 0.64. The highest fungal counts were recorded in fermented coconut with 7.4×102 sfu/g, while cocoa pod and groundnut shell had no fungal growth. In terms of percentage occurrence of fungal isolates from selected agrowastes, it was revealed that Rhizopus japonicus had the highest occurrence of 66.67%, while the same value of 8.33% was observed for Aspergillus fumigatus, A. glaucus, R. stolonifer and Trichosporonoides oedocephalis. In fermentation broth, all the 11 isolates displayed mannanase activity ranging from 0.370 to 21.667 U/ml for static and 0.278 to 3.982 U/ml for shaken condition, with the highest mannanase activity observed with isolate 5A for both culture conditions. According to the cultural characters and microscopic morphology, the isolate 5A being the highest mannanase producer was identified as the Aspergillus fumigatus. Conclusion: In this study, fungal isolates screened and evaluated for mannanase production from agricultural by-products elaborated considerable mannanase activity and this could be exploited for prebiotic preparation.

Fermenation Products of β-Mannanase Producing Bacteria Inhibit Selected Poultry Borne Pathogens.

Aim: The study evaluated the inhibitory effect of fermentation products of β-mannanaseproducing bacteria on selected poultry borne pathogens. Study Design: The first experiment, bacterial isolates previously confirmed positive for mannanase by plate assay technique were further screened for mannanase production in submerged state fermentation. In the second experiment, inhibitory effect of fermentation products of mannanase-producing bacteria on selected poultry pathogens was evaluated. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: Bacterial isolates from agricultural wastes previously confirmed positive for mannanase activity by plate assay were further screened for their potential performance under submerged state fermentation and enzyme activity determined by dinitrosalicylic acid method. The inhibitory action of β-mannanase-producing bacteria was determined by supplementation of supernatant and plating method. Results: Isolate 1A showed highest mannanase activity (13.430 U/ml), displayed broad inhibition to selected poultry borne pathogens; Klebsiella oxytoca, Shigella alkalescens, Escherichia coli, Salmonella typhii, Staphylococcus aureus and Streptococcus sp. Apart from isolate 1A, fermentation products of other isolates generated from the mannolytic action of β-mannanase on mannan containing substrate displayed different percentage inhibition on selected poultry borne pathogens. Conclusion: The results suggested that fermentation products from β-mannanaseproducing bacteria might possess antibacterial properties which could be applied in poultry farms.