scholarly journals Purification and Characterization of Aspartic Protease Produced from Aspergillus oryzae DRDFS13 under Solid-State Fermentation

2020 ◽  
Author(s):  
Jermen Mamo ◽  
Jorge Fernando Suarez Orellana ◽  
Vikas Yelemane ◽  
Martin Kangwa ◽  
Hector Marcelo Fernandez-Lahore ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Solid State ◽  
Ion Exchange ◽  
Solid State Fermentation ◽  
Aspartic Protease ◽  
Crude Enzyme ◽  
Crude Enzyme Extract ◽  
Standard Methods ◽  
Protease Enzyme ◽  
Cheese Production

AbstractAspartic proteases (E.C.3.4.23.) are endopeptidases with molecular masses ranging between 30–45 kDa. They depend on aspartic acid residues for their catalytic activity and show maximal activity at low pH. Thus the main objective of the present study was to purify and characterize aspartic protease from locally identified fungi by solid-state fermentation. The aspartic protease in the current study was obtained from A. oryzae DRDFS13 under SSF. The crude enzyme extract was purified by size-exclusion (SEC) and ion-exchange (IEC) chromatography. The protein contents of crude enzyme and IEC fractions were determined by BCA methods while the presence of N-glycosylation was checked using Endo-H. Inhibition studies were conducted using protease inhibitors. The milk-clotting activity (MCA), protease activity (PA); molecular weight and enzyme kinetics were determined using standard methods. Optimum temperature and stability, optimum pH and stability, and the effect of cations on MCA were assessed using standard methods. The maximum MCA (477.11 U/mL) was recorded from IEC fraction A8. The highest specific activity (183.50 U/mg), purification fold (6.20) and yield (9.2%) were also obtained from the same fraction (IEC A8). The molecular weight of 40 kDa was assigned for the purified enzyme (IEC A8). However, its molecular weight was decreased to 30 KDa upon deglycosylation assay which infers that the protein is glycosylated. Incubation of the pure enzyme (IEC A8) with pepstatin A caused a 94 % inhibition on MCA. The dialyzed enzyme showed a Km and Vmax values of 17.50 mM and 1369 U, respectively. The enzyme showed maximum MCA at 60 °C and pH 5.0 with stability at pH 4.5-6.5 and temperature 35-45 °C. Most cat-ions stimulate the activity of the enzyme; moreover, the highest MCA was detected at 50 mM of MnSO4. Furthermore, the results obtained in the present study confirmed that the aspartic protease enzyme produced from A. oryzae DRDFS13 and purified in ion-exchange chromatography could be used as a substitute source of rennet enzyme for cheese production.ImportanceThe production of pure aspartic protease enzyme from local microbes which is useful to substitute shortage of calf rennet enzyme and valuable to diversify cheese production throughout the world.

scholarly journals Kinetic Analysis of Crude Enzyme Extract Produced Via Solid State Fermentation of Banana Pseudo Stem Waste

2021 ◽  
Author(s):  
Mira Chares Subash ◽  
Muthiah Perumalsamy
Keyword(s):  
Solid State ◽  
Solid State Fermentation ◽  
Crude Enzyme ◽  
Enzyme Extract ◽  
Waste Biomass ◽  
Textile Processing ◽  
Banana Fiber ◽  
Crude Enzyme Extract ◽  
Banana Fibers ◽  
Pectinase Enzyme

Abstract Banana Pseudo stem waste after each harvest contributes about 70–80 Milli Tons Per hector. The banana pseudo stem will be thrown as waste biomass after each harvest as it is unstable for the upcoming harvest. The biggest challenge in banana cultivation is the utilization of biomass of banana pseusostem waste into valuable products. In this study, Xylano-pectinase enzyme extract was produced from banana pseudo stem waste under solid-state fermentation by Enterobacter cloacae PMC04. The highest pectinase and xylanase activities obtained using banana pseudo stem as carbon source were 124.62 U/ml and 173.81 U/ml respectively. Thermodynamics stated that range 40-50oC were considered to be the optimal temperature for xylano-pectinase enzyme production and subsequent degumming of banana fibers. The crude enzyme extract were then used in the degumming of banana fibers for textile application. Textile processing of banana fiber necessitates the removal of hemicellulose substance which can be achieved by crude xylano-pectinase enzyme. It was found that crude xylano-pectinase was efficient in the removal of hemicellulose substance from the fibers. Results obtained from this study demonstrate that the proposed bioprocess could be successfully applied for the degumming of banana fibers sustainably.

Kinetic Analysis of a Crude Enzyme Extract Produced via Solid State Fermentation of Bakery Waste

2015 ◽  
Vol 3 (9) ◽  
pp. 2043-2048 ◽  
Author(s):  
Wei Han ◽  
Wan Chi Lam ◽  
Mehmet Melikoglu ◽  
Man Tung Wong ◽  
Hoi Ting Leung ◽  
...  
Keyword(s):  
Solid State ◽  
Kinetic Analysis ◽  
Solid State Fermentation ◽  
Crude Enzyme ◽  
Enzyme Extract ◽  
Crude Enzyme Extract

scholarly journals Valorization of Lignocellulosic Wastes to Produce Phytase and Cellulolytic Enzymes from a -Thermophilic Fungus, Thermoascus aurantiacus SL16W, under Semi-Solid State Fermentation

2021 ◽  
Vol 7 (4) ◽  
pp. 286
Author(s):  
Keerati Tanruean ◽  
Watsana Penkhrue ◽  
Jaturong Kumla ◽  
Nakarin Suwannarach ◽  
Saisamorn Lumyong
Keyword(s):  
Solid State ◽  
Rice Bran ◽  
Solid State Fermentation ◽  
Reducing Sugar ◽  
Crude Enzyme ◽  
Enzyme Extract ◽  
Thermoascus Aurantiacus ◽  
Tea Leaves ◽  
Crude Enzyme Extract ◽  
Semi Solid

Agricultural wastes are lignocellulosic biomasses that contain high mineral and nutrient contents. This waste can be used as a raw material in industrial enzyme production by microbial fermentation. Phytase is an important enzyme used in animal feed to enhance the amount of phosphorus available for the growth and overall health improvement of monogastric animals. Fungi offer high potential as an effective source in the production of various extracellular enzymes. In this study, the production of lignocellulolytic enzymes (endoglucanase and xylanase) and phytase by a thermophilic fungus, namely Thermoascus aurantiacus strain SL16W, was evaluated using sixteen different Thai agricultural forms of waste under conditions of high temperature (45 °C). Semi-solid state fermentation was used in the production experiments. The results of this study reveal that the highest phytase activity (58.6 U/g substrate) was found in rice bran, whereas the highest degrees of activity of endoglucanase and xylanase were observed in wheat bran and red tea leaves at 19 and 162 U/g substrate, respectively. Consequently, the optimal conditions for phytase production of this fungus using rice bran were investigated. The results indicate that the highest phytase yield (58.6 to 84.1 U/g substrate) was observed in rice bran containing 0.5% ammonium sulfate as a nitrogen source with 10 discs of inoculum size at a cultivation period of 9 days at 45 °C and moisture content of 95%. Notably, the phytase yield increased by 1.71-fold, while endoglucanase and xylanase were also increased by 1.69- and 1.12-fold, respectively. Furthermore, the crude enzyme obtained from the optimal condition was extracted. The crude enzyme extract was then separately added to red tea leaves, rice straw, corncobs, palm residue, and peanut husks. Subsequently, total reducing sugar and phosphorus contents were determined. The results indicate that the highest level of reducing sugar (122.6 mg/L) and phosphorus content (452.6 mg/L) (p < 0.05) were obtained in palm residue at 36 and 48 h, respectively, after the addition of the crude enzyme extract. This study has provided valuable information on a potentially eco-friendly way to valorize agricultural waste into value-added products as industrial enzymes.

scholarly journals A Thermostable and Alkalitolerant Arabinofuranosidase by Streptomyces lividus

2020 ◽  
pp. 35-47
Author(s):  
Abimbola Olajide ◽  
Felicia C. Adesina ◽  
Abiodun A. Onilude
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Solid State ◽  
Solid State Fermentation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Palm Kernel ◽  
Residual Activity ◽  
Apparent Homogeneity ◽  
Production Temperature

Aim: The study aimed at producing and purifying thermostable and alkalitolerant microbial arabinofuranosidase using local Palm Kernel Cake (PKC) as substrate. Study Design: This is an experimental design in which samples were collected thrice and  subjected to laboratory analyses from which quantitative data were obtained and analysed. Place and Duration of Study: Ibadan, Nigeria, Five months. Methodology: Bacterial strains were isolated from degrading PKC by serial dilution and pour plate technique on formulated Modified Basal Salt Agar Medium and incubated at 50°C for enzyme activity screening. Plates were afterwards flooded with 1% congo red solution for visualization of hydrolysis zone. Its arabinofuranosidase activity was optimized in solid state fermentation in PKC. Production temperature, pH, moisture content, inoculum size and agitation were studied for optimization test. Optimal production temperature and pH for arabinofuranosidase by isolate was 45°C and pH 9. Produced arabinofuranosidase was purified to apparent homogeneity with ammonium sulphate precipitation, dialysis and column chromatography techniques. Stability of arabinofuranofuranosidase obtained to temperature, pH, substrate concentration and some ions was determined as well as its molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Isolate with highest arabinofuranosidase activity was selected and identified as Streptomyces lividus. Purity level attained was 16.36 fold. Enzyme had a specific activity of 25.4 U/mg, and total enzyme activity of 13.2 U.  Molecular weight of enzyme appeared as a band of 30 kDa. Purified arabinofuranosidase enzyme revealed optimum temperature and pH as 60oC and 9 respectively. Enzyme was stable over a broad pH range of 3-11, and temperature of 30-80oC. Residual activity after incubating for 1 hour at 70oC was 64%. Enzyme kinetics studies showed Km and Vmax values for P-nitrophenyl arabinofuranoside were 2.3mM and 0.7U/min respectively. Conclusion: Apart from Solid State Fermentation (SSF) of PKC being a potential fermentation technique for production of arabinofuranosidase by Streptomyces lividus, the enzyme was highly stable.

scholarly journals Growth and cutinase activity of Fusarium culmorum grown in solid-state fermentation

2016 ◽  
Vol 1 (2) ◽  
pp. 8-19 ◽  
Author(s):  
Miriam Sofía Canavati-Alatorre ◽  
Israel Águila ◽  
Ilse Karina Barraza-Soltero ◽  
Elizabeth Castillón ◽  
Ana Laura Correa-Barrón ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Solid State ◽  
Carbon Source ◽  
Solid State Fermentation ◽  
Food Industry ◽  
Culture Media ◽  
Fusarium Culmorum ◽  
Glucose Consumption ◽  
Toxic Substances ◽  
Wide Range

Cutinase is a very versatile enzyme due to the wide range of substrates that it can use. It has application in several biotechnological areas, acting as biocatalysts in the food industry, in detergents as well as in biodegradation of polymers and other toxic substances. In this research, glucose consumption, protein content, biomass and cutinase production by Fusarium culmorum were evaluated. Cutinolitic activity was observed using gel zymography. This fungus was grown on culture media containing glucose and different concentrations of apple cutin (0, 1, 3 and 10 g/L) in solid-state fermentation. F. culmorum showed the highest production of biomass, protein and cutinase in medium supplemented with 10 g of cutin/L. Glucose uptake was inversely proportional to the cutin concentration. Medium lacking cutin showed the fastest glucose consumption. Heavily stained bands were observed in the gel with increasing concentrations of cutin after 72 h, showing a molecular weight of 50 kDa. These results shown that apple cutin induced cutinase production and was also used as carbon source. It was also observed that glucose did not act as a repressor of cutinase production.

scholarly journals Solid State Fermentation of a Raw Starch Digesting Alkaline Alpha-Amylase from Bacillus licheniformis RT7PE1 and Its Characteristics

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Romana Tabassum ◽  
Shazia Khaliq ◽  
Muhammad Ibrahim Rajoka ◽  
Foster Agblevor
Keyword(s):  
Molecular Weight ◽  
Solid State ◽  
Bacillus Licheniformis ◽  
Wheat Bran ◽  
Solid State Fermentation ◽  
Half Life ◽  
Alpha Amylase ◽  
Kinetic Properties ◽  
Optimum Temperature ◽  
Raw Starch

The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacillus licheniformis RT7PE1 strain were studied. The kinetic values Qp, Yp/s, Yp/X, and qp were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112 kDa. The apparent Km and Vmax values for starch were 3.4 mg mL−1 and 19.5 IU mg−1 protein, respectively. The optimum temperature and pH for α-amylase were 55°C, 9.8. The half-life of enzyme at 95°C was 17h. The activation and denaturation activation energies were 45.2 and 41.2 kJ mol−1, respectively. Both enthalpies (ΔH∗) and entropies of activation (ΔS∗) for denaturation of α-amylase were lower than those reported for other thermostable α-amylases.

scholarly journals Production of ethanol and clarification of apple juice by pectinase enzyme produced from Aspergillus terreus NCFT 4269.10

2016 ◽  
Vol 4 (1) ◽  
pp. 67 ◽  
Author(s):  
Bijay Sethi ◽  
Amrita Satpathy ◽  
Subodh Tripathy ◽  
Sidarth Parida ◽  
Sameer Kumar Singdevsachan ◽  
...  
Keyword(s):  
Solid State ◽  
Wheat Bran ◽  
Solid State Fermentation ◽  
Apple Juice ◽  
Aspergillus Terreus ◽  
Specific Activity ◽  
Sole Source ◽  
Crude Enzyme ◽  
Maximum Production ◽  
Pectinase Production

Aspergillus terreus NCFT 4269.10 was evaluated by liquid static surface fermentation (LSSF), shaking fermentation (ShF) and solid state fermentation (SSF) for the production of pectinase. Among various substrates tested, banana peels supported maximum production of pectinase i.e. 1000 ± 141.42 U/ml. The biomass of A. terreus was maximum with wheat bran (0.55±0.07g/50ml). Pectinase produced by A. terreus displayed higher specific activity when wheat bran was used as the sole source of carbon and energy. After successful fermentation, crude enzyme was purified to electrophoretic homogeneity with a specific activity of 1846.50 U/mg from an initial specific activity of 1282.05 U/mg. The cell free-dialyzed-enzyme containing 107100 U was purified to 1.44 fold with an overall enzyme yield of 35.70%.Immobilization study revealed that the production of pectinase was increased up to third cycle and decreased thereafter when further pectinase production was carried out by immobilized spores of A. terreus.

scholarly journals Evaluasi Perlakuan Pendahuluan Menggunakan Kalsium Hidroksida untuk Biokonversi Jerami Padi Menjadi L-Asam Laktat oleh Rhizopus oryzae AT3 (Evaluation of Lime Pretreatment for Bioconversion of Rice Straw to L-Lactic Acid by Rhizopus Oryzae AT3)

2016 ◽  
Vol 36 (03) ◽  
pp. 253
Author(s):  
Dhina Aprilia Nurani Widyahapsari ◽  
Retno Indrati ◽  
Sigit Setyabudi ◽  
Sardjono Sardjono
Keyword(s):  
Lactic Acid ◽  
Solid State ◽  
Enzymatic Hydrolysis ◽  
Rice Straw ◽  
Solid State Fermentation ◽  
Reducing Sugars ◽  
Rhizopus Oryzae ◽  
Crude Enzyme ◽  
Lime Pretreatment ◽  
Rice Straw Hydrolysate

L-lactic acid can be used as a precursor of polylactic acid (PLA). PLA is a biodegradable biomaterial commonly used for biodegradable plastics. Lactic acid can be produced from lignocelluloses materials such as rice straw. Rice straw is composed of cellulose and hemicellulose that can be hydrolyzed to fermentable sugar by cellulolytic and hemicellulolytic enzymes then converted to L-lactic acid by Rhizopus oryzae. As most cellulose and hemicellulose present in lignocellulose biomass are not readily accessible for these enzyme, pretreatment is required to alter the structure of lignocellulose substrates. This research aimed to investigate the effect of lime pretreatment on rice straw bioconversion to L-lactic acid by Rhizopus oryzae AT3. Rice straw was pretreated with lime (Ca(OH)2) at 85 °C for 16 hours. Unpretreated and pretreated rice straw were hydrolyzed using crude enzyme that produced by Trichoderma reesei Pk1J2. Enzyme production was carried out by solid state fermentation using rice straw and rice brand as substrate. Enzymatic hydrolysis was carried out in flasks. Each flask was added with unpretreated or pretreated rice straw, buffer citrate solution and crude enzyme then hydrolyzed for 0-96 hours. Hydrolysate was fermented by Rhizopus oryzae AT3 for 0-6 days by using adsorbed carrier solid-state fermentation method with polyurethane foam as inert support material. Lime pretreatment at 85 °C for 16 hour led to significant solubilisation of lignin and hemicellulose. It involved lignocellulose structure modified that enhance enzymatic hydrolysis and resulted higher reducing sugars than unpretreated rice straw. The high reducing sugars was not related to high lactic acid yields. Fermentation of pretreated rice straw hydrolysate by Rhizopus oryzae AT3 did not only produce L-lactic acid but also other compound. On the other hand, fermentation of unpretreated rice straw hydrolysate only produced L-lactic acid. ABSTRAKPolimerisasi asam laktat menjadi polylactic acid untuk menghasilkan biodegradable plastic membutuhkan asam laktat dengan isomer spesifik. Rhizopus oryzae adalah mikroorganisme yang spesifik menghasilkan L-asam laktat. Selain itu Rhizopus oryzae dapat menggunakan limbah pertanian seperti jerami padi sebagai substrat. Komponen utama jerami padi merupakan lignoselulosa yang dapat dihidrolisa secara enzimatis menjadi komponen gula sederhana penyusunnya dan selanjutnya dapat dikonversi menjadi L-asam laktat oleh Rhizopus oryzae. Namun struktur lignoselulosa sangat kompak dan rapat, sulit untuk dihidrolisa secara enzimatis sehingga diperlukan adanya perlakuan pendahuluan untuk merombak struktur lignoselulosa agar mudah dihidrolisa. Penelitian ini bertujuan untuk mengetahui pengaruh perlakuan pendahuluan menggunakan kalsium hidroksida (Ca(OH)2) terhadap biokonversi jerami padi menjadi L-asam laktat oleh Rhizopus oryzae AT3. Perlakuan pendahuluan pada jerami padi dilakukan menggunakan (Ca(OH)2) disertai pemanasan suhu 85 °C selama 16 jam. Jerami padi dengan dan tanpa perlakuan pendahuluan dihidrolisa secara enzimatis menggunakan crude enzyme yang diproduksi oleh Trichoderma reesei Pk1J2. Produksi crude enzyme dilakukan dengan fermentasi substrat padat dengan campuran jerami padi dan dedak sebagai substrat. Hidrolisat jerami padi dengan dan tanpa perlakuan pendahuluan selanjutnya difermentasi oleh Rhizopus oryzae AT3 menggunakan metode adsorbed carrier solid state fermentation dengan polyurethane foam (PUF) sebagai bahan pendukung. Perlakuan pendahuluan menggunakan Ca(OH2) disertai pemanasan suhu 85 °C selama 16 jam dapat merubah komposisi lignoselulosa jerami padi yaitu dengan melarutkan lignin dan hemiselulosa. Perubahan komposisi lignoselulosa memudahkan kerja crude enzyme dalam menghidrolisa jerami padi sehingga menghasilkan gula reduksi lebih tinggi dibandingkan jerami padi tanpa perlakuan pendahuluan. Tingginya gula reduksi tidak serta merta meningkatkan yield L-asam laktat yang dihasilkan. Fermentasi hidrolisat jerami padi dengan perlakuan pendahuluan oleh Rhizopus oryzae AT3 menghasilkan yield L-asam laktat lebih rendah dibandingkan hidrolisat jerami padi tanpa perlakuan pendahuluan. Namun pada jerami padi dengan perlakuan pendahuluan dihasilkan senyawa lain selain asam laktat.Kata Kunci: Adsorbed carrier solid state fermentation; L-asam laktat; perlakuan pendahuluan; Rhizopus oryzae AT3; jerami padi

Purification and Characterization of Aspartic Protease Produced from Aspergillus oryzae DRDFS13 MN726447 under Solid-State Fermentation

2021 ◽  
Author(s):  
Jermen Mamo ◽  
Martin Kangwa ◽  
Jorge Fernando Suarez Orellana ◽  
Vikas Yelemane ◽  
Hector Marcelo Fernandez-Lahore ◽  
...  
Keyword(s):  
Solid State ◽  
Aspergillus Oryzae ◽  
Solid State Fermentation ◽  
Aspartic Protease ◽  
Purification And Characterization
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