An overview of the solid state fermentation in the production of fungal protease enzymes

Enzymes are among the most important products obtained for human needs through microbial sources. A large number of industrial processes in the area of industrial, environmental and food technology utilize enzymes at some stage or another; Solid State Fermentation (SSF) holds tremendous potential for the production of enzymes, especially in those processes where the crude fermented product may be used directly as the enzyme source. Fungal proteases are used in many industrial processes for the production of foods and metabolites, production of enzymes from fungi offered many advantages which include low cost and high productivity. Hence because of the higher yielding capacity of SSF and the demand for proteases it highly imperative to search for novel microorganisms from possible environment and subject them to SSF for protease investigation to add up to the nation need of the enzymes and boast economy.

Degradation of Wheat Germ Agglutinin during Sourdough Fermentation

Non Celiac Wheat Sensitivity (NCWS) is an intolerance to wheat products and individuals with NCWS often adhere to a gluten free diet. However, gluten free diets are often associated with a reduced sensory and nutritional quality. Wheat Germ Agglutinin (WGA) is one of the wheat components linked to NCWS. This study explored the fate of WGA during sourdough fermentation. To assess the role of thiol-exchange reactions and proteolysis, sourdoughs were fermented with Fructilactobacillus sanfranciscensis DSM20451, F. sanfranciscensis DSM20451ΔgshR, which lacks glutathione reductase activity, or Latilactobacillus sakei TMW1.22, with or without addition of fungal protease. The conversion of WGA was determined by size exclusion chromatography of fluorescence-labeled WGA, and by enzyme-linked immunosorbent assay (ELISA). Commercial whole wheat flour contained 6.6 ± 0.7 μg WGA/g. After fermentation with L. sakei TMW1.22 and F. sanfranciscensis DSM20451, the WGA content was reduced (p < 0.05) to 2.7 ± 0.4 and 4.3 ± 0.3 μg WGA/g, respectively, while the WGA content remained unchanged in chemically acidified controls or in doughs fermented with F. sanfranciscensis DSM20451ΔgshR. Protease addition did not affect the WGA content. In conclusion, the fate of WGA during sourdough fermentation relates to thiol-exchange reactions but not to proteolytic degradation.

Fibrinogenolytic activity of protease from the culture fluid of Pleurotus ostreatus

The use of proteases makes it possible to obtain partially hydrolyzed forms of macromolecules with unique properties. The importance of proteases for studying the structure and functions of fibrinogen forces scientists to search for new sources of highly specific proteases. Thus, the aim of this work was to study the content of the Pleurotus ostreatus culture fluid in search of fibrinogen- specific proteases. P. ostreatus was cultured for 14 days at 27°C. The culture fluid was collected and the protein fraction was salted out with NaCl and then dialyzed. Fibrinogen hydrolysis products by P. ostreatus protease were characterized using SDS PAGE under reducing conditions followed by immunoprobing using murine monoclonal antibodies I-5A (anti-Aα505-610) and 2d2a (anti-Bβ26-42). The study of turbidity and platelet aggregation was performed using a Multiskan FC spectrophotometric microplate reader and a SOLAR-2110 aggregometer, respectively. Electron microscopy of fibrils formed by truncated compared with native fibrins was performed using a transmission electron microscope N-600. Analysis of the products of fibrinogen hydrolysis with a fungal protease using SDS-PAGE demonstrated the cleavage of the alpha chain of fibrinogen exclusively with the formation of a truncated form of fibrinogen in which there are no C-terminal portions of αC regions with a molecular weight of 25 kDa. A study of turbidity showed that the polymerization of truncated fibrin is significantly impaired. The rate of lateral association of protofibrils significantly decreased from 1.5 to 2.2 times in the case of truncated fibrinogen compared to the native one depending on the initial concentration of fibrinogen. It was shown that platelet aggregation in the presence of fibrinogen without 25 kDa fragments of αC regions was less effective than in the presence of native fibrinogen. Application of the preparation of the fungal protease allows us to obtain high molecular forms of the fibrinogen molecule with cleaved 25 kDa peptides, which provide new information on the role of these peptides in the fibrinogen functioning.

Fibrin(ogen)olytic and platelet modulating properties of a novel protease from the culture fluid of Pleurotus osreatus

The use of proteases makes it possible to obtain partially hydrolyzed forms of macromolecules with unique properties. The importance of proteases for studying the structure and functions of fibrinogen forces scientists to search for new sources of highly specific proteases. Thus, the aim of this work was to study the content of the Pleurotus ostreatus culture fluid in search of fibrinogen-specific proteases. P. ostreatus was cultured for 14 days at 27 ° C. The culture fluid was collected and the protein fraction was salted out with NaCl and then dialyzed. Fibrinogen hydrolysis products by P. ostreatus protease were characterized using SDS PAGE under reducing conditions followed by immunoprobing using murine monoclonal antibodies I-5A (anti-Aα505-610) and 2d2a (anti-Bβ26-42). The study of turbidity and platelet aggregation was performed using a Multiskan FC spectrophotometric microplate reader and a SOLAR-2110 aggregometer, respectively. Electron microscopy of fibrils formed by truncated compared with native fibrins was performed using a transmission electron microscope N-600.Analysis of the products of fibrinogen hydrolysis with a fungal protease using SDS-PAGE demonstrated the cleavage of the alpha chain of fibrinogen exclusively with the formation of a truncated form of fibrinogen in which there are no C-terminal portions of αC regions with a molecular weight of 25 kDa. A study of turbidity showed that the polymerization of truncated fibrin is significantly impaired. The rate of lateral association of protofibrils significantly decreased from 1.5 to 2.2 times in the case of truncated fibrinogen compared to the native one depending on the initial concentration of fibrinogen. It was shown that platelet aggregation in the presence of fibrinogen without 25 kDa fragments of αC regions was less effective than in the presence of native fibrinogen. Application of the preparation of the fungal protease allows us to obtain high molecular forms of the fibrinogen molecule with cleaved 25 kDa peptides, which provide new information on the role of these peptides in the fibrinogen functioning.

Combined ANN/ EVOP Factorial Design Approach for Media Screening for Cost-effective Production of Alkaline Proteases from Rhizopus oryzae (SN5) / NCIM-1447 under SSF

In order to achieve high yield of fungal protease in a very cost effective way and to meet its increased market demand, current study deals with the screening of various agro-wastes as carbon source for the production of protease from Rhizopus oryzae (SN5) / NCIM-1447 under solid state fermentation. Substrates and culture parameters such as wheat bran, soybean meal, black-gram husk, rice husk, mixture of wheat bran, soybean meal, nitrogen sources, pH, temperature and incubation time were first optimized with one factor at time strategy and then EVOP factorial and yield of alkaline protease was achieved 412.8 U/gds at 28 o C and pH=6 after 72 hours of fermentation taking wheat bran and soybean as a substrate in 4:1 ratio. Further Artificial Neural Networks (ANN), was trained with data of EVOP and yield of protease was enhanced up to 422.6 U/gds with wheat bran: soyabean in ratio of 70:30, pH 6.2 at 30°C. The evolved process and Rhizopus oryzae (SN5)/ NCIM-1447 strain would be promising for protease production at industrial scale at low cost.

Epithelial damage triggers allergic inflammation

A fungal protease induces allergic inflammation by damaging the lung epithelium.

Combined ANN/ EVOP Factorial Design Approach for Media Screening for Cost-effective Production of Alkaline Proteases from Rhizopus oryzae (SN5) / NCIM-1447 under SSF

In order to achieve high yield of fungal protease in a very cost effective way and to meet its increased market demand, current study deals with the screening of various agro-wastes as carbon source for the production of protease from Rhizopus oryzae (SN5) / NCIM-1447 under solid state fermentation. Substrates and culture parameters such as wheat bran, soybean meal, black-gram husk, rice husk, mixture of wheat bran, soybean meal, nitrogen sources, pH, temperature and incubation time were first optimized with one factor at time strategy and then EVOP factorial and yield of alkaline protease was achieved 412.8 U/gds at 28 o C and pH=6 after 72 hours of fermentation taking wheat bran and soybean as a substrate in 4:1 ratio. Further Artificial Neural Networks (ANN), was trained with data of EVOP and yield of protease was enhanced up to 422.6 U/gds with wheat bran: soyabean in ratio of 70:30, pH 6.2 at 30°C. The evolved process and Rhizopus oryzae (SN5)/ NCIM-1447 strain would be promising for protease production at industrial scale at low cost.

Combined ANN/ EVOP Factorial Design Approach for Media Screening for Cost-effective Production of Alkaline Proteases from Rhizopus oryzae (SN5) / NCIM-1447 under SSF

In order to achieve high yield of fungal protease in a very cost effective way and to meet its increased market demand, current study deals with the screening of various agro-wastes as carbon source for the production of protease from Rhizopus oryzae (SN5) / NCIM-1447 under solid state fermentation. Substrates and culture parameters such as wheat bran, soybean meal, black-gram husk, rice husk, mixture of wheat bran, soybean meal, nitrogen sources, pH, temperature and incubation time were first optimized with one factor at time strategy and then EVOP factorial and yield of alkaline protease was achieved 412.8 U/gds at 28oC and pH=6 after 72 hours of fermentation taking wheat bran and soybean as a substrate in 4:1 ratio. Further Artificial Neural Networks (ANN), was trained with data of EVOP and yield of protease was enhanced up to 422.6 U/gds with wheat bran: soyabean in ratio of 70:30, pH 6.2 at 30°C. The evolved process and Rhizopus oryzae (SN5)/NCIM-1447 strain would be promising for protease production at industrial scale at low cost.