Submerged Fermentation System Research Articles

Bio-hydrolysis Study of Lactose in Produced Yoghurt by β-Galactosidase from Lactobacillus fermentum

The present study investigated the physicochemical and biohydrolysis of lactose in conventional and locally produced yoghurt drinks, respectively. Physicochemical properties of the conventional yoghurt in the presence of the locally produced one were: pH 3.0; 4.0, titratable acid 1.14; 1.04, conductivity 2751.54, 2204, viscosity 71.11; 112.14, TDS 1788.5; 1433, TSS 781; 654, TS 3532.54; 2087, Mg 11.06; 34.19, Ca 28.76; 45.21, TOC 155.23; 145.23 and TOM 190.93, 178.63 for yoghurt sample I and II respectively. Lactose concentrations of the respective yoghurts were: 2.12 and 1.79 Mg/ml. β-galactosidase was produced from isolated Lactobacillus sp under submerged fermentation system after 12 days of incubation. 60% ammonium sulphate was found suitable for precipitation of proteins with highest enzyme activity, the precipitate was desalted through reverse osmosis using dialysis bag. Optimum biohydrolysis was achieved after 20 hours of incubation with residual sugar concentration of 0.51 mg/ml while the control experiment had 0.93 mg/ml of the residual sugar. Optimum pH and temperature of hydrolysis were: 5.0 and 70°C respectively. Rate of hydrolysis proceed linearly as the enzyme concentration increases from 0-20% v/v. the present study has shown exclusively the nutritional components of yoghurts drinks commercially sold in the market, it went further to show hope for the metabolic compromised individuals with intolerance to lactose as the enzyme lactase produced from non noscomial organism showed great potentials of hydrolysis.

Enhancement of Anti-MRSA Potential Produced by an Endophytic Fungus Ceratobasidium Ramicola IBRLCM127 via Submerged Fermentation System

Introduction: Exploring endophytic fungi isolated from medicinal herbs could be a turning point in the research of secondary metabolites biosynthesis, as these endophytic fungi are capable of synthesizing the similar compounds as their host plant. The advantages of manipulating endophytic fungi for bioactive compound production are the reduction of dependency rate on slow-growing and rare plants, cost-effective, continuous process, environmentally friendly and high yield in a short period. Thus, the current study envisages investigating the influence of culture conditions against the anti-MRSA potential production of the endophytic fungal isolate, Ceratobasidium ramicola IBRLCM127 isolated from the local medicinal plant Curcuma mangga Valeton & Zijp. Methods: The endophytic fungal isolate was used to produce fungal metabolites through submerged fermentation. The physical parameter improvement was investigated using the ‘one-factor-at-a-time’ technique. The fungal fermentative broth was subjected to an anti-MRSA assay using Lorian method, whereas the growth of a fungus was determined based on the cell growth weight. Results: The highest anti-MRSA potential of 42.50±0.1 U/ml and 5.49±0.1 g/L of mycelial growth was observed after improving the basal medium containing yeast extract sucrose broth incorporated with water extract from the host plant, 6 days old of inoculum age, 2 agar plugs of mycelia, incubation temperature of 25 0C and 12 days of cultivation 12 days of cultivation shaken at 120 rpm in the absence of light. Conclusion: The improved culture conditions shorten the incubation period and yield a significant enhancement of anti-MRSA potential and fungal growth with 13.27% and 10.91%, respectively.

Screening of Conditions for Manganese Peroxidase Production from White Rot Fungi (Pleurotus djamor)

The need for alternative and environmentally friendly methods for waste clean-up has led to the use of enzymes in bioremediation. In this study, white rot fungi were isolated from decaying plant parts using standard microbiological and biochemical techniques. The isolated fungi were identified and screened using standard substrates (2, 6-DMP) to determine their capability to produce manganese peroxidase. Pure manganese peroxidase was achieved after four distinct purification phases. Crude extract of the homogenate proteins obtained through optimized submerged fermentation system were precipitated using ammonium sulfate. Ammonium sulfate used at 60% concentration at pH 4.5 precipitated proteins with highest Manganese peroxidase activity (322 U/mg). The precipitated proteins were desalted through dialysis for twelve hours with buffer exchange at interval of six hours and activity of 343.91 U/mg was recorded afterwards. DEAE-cellulose was used for the ion exchange purification of the dialyzed protein, NaCl gradients of 0.3-0.6 M was found to be best in washing off bound protein from the exchange resin and activity of 434.18 U/mg was recorded from the pooled fraction tubes. Sephadex G-100 was used for separation of the proteins into molecular sizes and weights. 2.8 purification folds of the enzyme were achieved after ion exchange (DEAE-cellulose) and gel filtration (sephadex G-100) with percentage yield of 2.20%. Specific activity of the enzyme increased to 602.00% after gel filtration. The partial purified enzyme was further characterized for determination of optimal pH (4.5), temperature (40 OC) and kinetic properties Km of 3.4 mM and Vmax of 250µmol/min.

Stain removing, juice-clarifying, and starch-liquefying potentials of amylase from Pleurotus tuberregium in submerged fermentation system

BackgroundAmylase is used commercially in food, textiles, sugar syrup, paper, and detergent industries. Bacteria and fungi remain a significant source of industrial enzymes. Pleurotus tuberregium is a macro-fungi that can exist as a fruiting body, sclerotium, mycelium, and spores. Some studies have been conducted on this fungus, with minimal studies on its enzyme activity (s) using the submerged fermentation technique. ResultsThe purified amylase has a specific activity of 5.26 U/mg, total activity of 189.20 U, maximally active at 70 °C, pH of 5, and retaining 100% of its activity at 30 oC for 4 min. P. tuberregium amylase showed optimal activity with plantain peel, followed by starch and pineapple peel (42, 30, and 29 μg/mL/min respectively). The presence of Ca2+, Mg2+, and Na+ ions in the reaction mixture activated the enzyme activity, but was slightly and moderately inhibited by KCl and Na2H2PO4 respectively. The crude enzyme effectively clarified juice, liquefied soluble cassava starch (with a release of appreciable glucose quantity), and partially de-stained white fabric. ConclusionsThe amylase obtained from the submerged fermentation of Pleurotus tuberregium has potential applications in food and detergent industries.

Studies on Properties of Lipase Produced from <i>Aspergillus</i> sp. Isolated from Compost Soil

Lipase producing Aspergillus was isolated from soil collected from a refuse dump site located at Awka, Anambra State, Nigeria using standard microbiology and biochemical techniques. Crude extract of lipase was produced after a successful screening of the isolates using mineral broth containing p-NPP through submerged fermentation system with optimized physiologic conditions. Three steps of purification were carried out: Ammonium sulphate, dialysis and gel filtration (sephadex G-150). Crude extract was precipitated by using 70% saturation of ammonium sulphate at pH 6.0 which gave the optimum precipitation of the protein with specific activity of 260.56 U/mg. Precipitation using ammonium sulphate carried out at pH 6.5 and 8.0 gave specific activity of 217 U/mg of the protein. The precipitates were further desalted through dialysis for twelve hours and specific activity of 343.20 U/mg was recorded from the dialysate afterwards. Further purification was done by using sephadex G-150 and specific activity of 490.55 U/mg was recorded from the active pooled fractions. The purification table showed a 2.32 purification folds of lipase was gotten after gel filtration (sephadex G-150) with a lipase percentage yield of 2.00%. The specific activity of lipase increased from 211.81 to 490.55 U/mg. Characterization of β-galactosidase gave optima pH and temperature of the enzyme at 6.0 and 60°C respectively. Kinetic constants: Km and Vmax values were obtained at various concentrations of p-NPP where 0.32 mM and Vmax of 200.00 μmol/min respectively. Ca2+ and Co2+ showed greater effect on lipase activity in a concentration-dependent manner (0.03 - 0.05 M) when compared to Mn2+ and Fe2+. The results from this study have shown that lipase produced from filamentous Aspergillus has a wide range of activity over physiologic conditions in regards to industrial and clinical standard operational procedures.

Production and characterization of cellulase from mushroom (Pleurotus ostreatus) for effective degradation of cellulose

Cellulases are a group of hydrolytic enzymes capable of hydrolyzing the most abundant organic polymer that means cellulose to smaller sugar components including glucose subunits. The aim of this study was to screen cellulase producing oyster mushroom collected from Eucalyptus tree bark to evaluate the in vitro production of cellulase by Pleurotus ostreatus using different lignocellulosic substrates, and to characterize the cellulase produced with respect to changes in pH, temperature, and concentration of substrates. A total of ten mushroom specimens were randomly collected from Eucalyptus tree bark in the premise of Holetta Agricultural Research Center campus. All of the collected mushroom specimens were identified morphologically and biochemically as Pleurotus ostreatus and also screened for their ability to produce cellulase by detecting and measuring zone of hydrolysis on commercial media containing Carbxymethyl Cellulose (CMC) as the sole carbon source. These mushroom specimens were cultivated using both solid state fermentation and submerged fermentation systems supplemented with different lignocellulosic substrates (wheat straw, teff straw, bean straw, wood fiber and Eucalyptus tree bark) to identify the most suitable medium for the production of cellulase. The highest enzyme production was obtained on bean straw and wheat straw which resulted in 0.191 U/ml, 0.868 U/ml and 0.389 U/ml; and 0.216 U/ml, 0.444 U/ml, and 0.245 U/ml of FPase, CMCase, and β-glucosidase in solid state fermentation. The lowest values were, however, obtained in media containing wood fiber in both solid state fermentation and submerged fermentation. Comparison of the lignocellulosic substrates revealed that wheat straw was selected for further growth parameter optimization. The production of cellulase was higher at the 5th day of incubation period, and the optimum pH and incubation temperature required for maximum cellulase production were 4 and 30°C, respectively. Sucrose and Yeast extract at 1% concentration were found to be the most preferred carbon and nitrogen sources for cellulase production by Pleurotus ostreatus. The optimum pH and temperature for cell_free cellulase activity on were found to be 4 and 50°C, respectively. Generally the cellulases produced by Pleurotus ostreatus were stable and active at temperatures ranging from 20-50°C. These characteristics hopefully would make this enzyme potentially attractive in a variety of industrial applications including animal feed treatments. There was a linear relationship between cellulase and its substrate concentration for there was an increase in activity with increase in substrate concentration. The relationship between rate of reaction and substrate concentration depended on the affinity of the enzyme for its substrate. Finally the cellulase was tested for its ability to saccharify agricultural wastes and the results showed the highest release of sugars from wheat straw.

A review of the use of microbial amylase in industry

Amylases are one of the most often utilized enzymes in business. Enzymes like these hydrolyze starch molecules into polymers made up of glucose units. Amylases have the potential to be used in a variety of commercial activities, including food, fermentation, and pharmaceuticals. Amylases come from a variety of sources, including plants, animals, and microbes. In the industrial sector, however, enzymes derived from fungi and bacteria have prevailed. The enzyme amylase is required for the conversion of starches to oligosaccharides. Starch is a key component of the human diet and a significant storage product in a variety of commercially important crops, including wheat, rice, maize, tapioca, as well as potato. Maltodextrin, modified starches, and glucose or fructose syrups are all made using starch converting enzymes. A wide range of microbial amylases are used in a variety of industries, including food, textiles, paper, and detergents. Submerged fermentation has traditionally been used to make amylases, however submerged fermentation systems seem to be a viable technique. The thermo stability, pH profiles, pH stability, and Ca-independency of each amylase are essential in the development of the fermentation process. The synthesis of bacteria and fungi amylases, their distribution, structural-functional features, physical and chemical parameters, and their usage in industrial applications are all covered in this study.

Exploring Simplified Methods for Insect Chitin Extraction and Application as a Potential Alternative Bioethanol Resource.

Simple SummaryThe studies on chitin utilization as a source for bioethanol production are still very few. The present study explores some simple methods for insect chitin extraction and application in bioethanol production. Using insect chitin in bioethanol production, may help decreasing the dependence on energy crops as a carbon source for bioethanol. Fungal strains of Mucor circinelloides were reported previously to bio-convert chitin directly to ethanol in submerged fermentation systems. In our study, we explored the bioconversion of insect chitin to bioethanol using two different strains of Mucor circinelloides in submerged fermentation systems. An insect-isolated M. circinelloides strain was found to bio-convert the extracted chitin directly to ethanol in submerged fermentation system. The source of strain isolation and the pH of the production medium were showed to influence the chitin bioconversion directly to bioethanol. All fermentation processes can be conducted easily, using the whole growing microorganism instead of using purified enzymes. These results highlight the insect biomass as a potential new, cheap and renewable source for bioethanol production simply, using a potent insect-isolated M. circinelloides strain.Chitin, the second most plentiful biopolymer in nature, is a major component of insect cuticle. In searching for alternative resources for fossil fuels, some fungal strains of Mucor circinelloides from an insect-source were found to produce bioethanol directly using insect chitin as a substrate. Herein, simplified methods for insect chitin extraction and application as a substrate in submerged fermentation for bioethanol production were explored. Chitin of the American cockroach (Periplaneta americana (L.)) was isolated by refluxing the cockroaches dried exoskeletons with 4% NaOH. The purity of the extracted chitin was assessed to be high when the physicochemical properties of the extracted chitin matched these of commercially available crab and shrimp samples. The extracted chitin was employed as a substrate in submerged fermentation using two strains of M. circinelloides. One of these, strains M. circinelloides 6017 showed immense potential for bioethanol production directly. It could to bio-transform 15 g/L of colloidal chitin directly to 11.22 ± 0.312 g/L of bioethanol (74% of the initial chitin mass) after 6 days of incubation. These results confirm the possibility of using insect biomass as a potential alternative resource for bioethanol production in a simple manner thus contributing to the creation of an alternate energy source.

A NEW TRIAL FOR BIOFORMATION OF ANTIMICROBIAL AGENT CONTROLLING MULTI DRUG RESISTANT MICROORGANISM

In the present investigation, a trial was done to find a new antimicrobial agent producing microbe from soil microbiota of local habitats to control the problem of multiple drug resistance. The term of Antimicrobial resistance (AMR) is used to describe microorganisms that can resist the effects of drugs and chemicals designed to kill them. Seventy six actinomycetes isolates were isolated from fifteen soil samples different localities in Egypt were primary screening for antimicrobial activity by agar plug diffusion method against test microorganisms. Sixteen isolates were selected for secondary screening in small scale submerged fermentation system and assayed against pathogenic tested microorganisms using agar well diffusion method. Among of these isolates tested, the isolate (S1SHA1) showed the highest antimicrobial activity against pathogenic test organisms. This isolate was identified as Streptomyces griseoplanus by morphological, physiological, biochemical characters and 16s rRNA gene sequence. Physical and nutritional factors affecting activity of antimicrobial agent were studied. The results showed that, optimum activity of antimicrobial agent achieved with pH 7, incubation temperature 28 C, for 7 days, at 150 rpm agitation, carbon and nitrogen source starch 1.5% and potassium nitrate 0.4%, as well as phosphorus 2 g/l and NaCl at concentration of 1 %. Antimicrobial agent from batch culture was subjected to extraction and purification processes using ethyle acetate and preparative TLC, respectively. Determination of minimum inhibitory concentrations (MIC) and Mode of action of antimicrobial agent produced by S. griseoplanus (S1SHA1) on the test microbial strains using Transmission Electron Microscopy (TEM). Cytotoxic studies showed that no cytotoxic effects were observed for the compound when tested even at high concentrations

Study of Ganoderma lucidum in Laccase Production using Corncob and Paddies Straw Substrates on Submerged Fermentation System.

Ganoderma lucidum a white rot fungi, produce laccase which capable to degrade lignin due to its activity as ligninolytic enzymes. The production of laccase by G. lucidum using various agroindustrial wastes, including corncob and paddies straw, as substrates have been studied. The purpose of this study was to determine substrate that able to produce the highest activity of the laccase from the G. lucidum. The method used an experimental design followed by descriptive analysis using 4 treatments with duplication including treatment G. lucidum growth into A (control, Potato Dextrose Broth (PDB)), B (PDB+corncob), C (PDB+rice straw) and D (PDB+corncob+rice straw). This study includes; (1) Qualitative assay determination of laccase, (2) Extraction and laccase activity, (3) Cell concentration of G. lucidum measurement and (4) pH measurement. Laccase qualitative assay showed brownish red ring on PDA media that indicated a positive of the laccase enzyme secreted by G. lucidum. Enzyme activity under submerged fermentation condition was achieved by the treatment of adding corncobs with the highest activity accounting 68.75 U mL-1. The fermentation process causes a decrease in pH during the incubation time to pH 4.83. The results of pH measurements showed that the laccase enzyme from G. lucidum worked optimally at pH 4-5 achieved after 5 day of incubation. Our results suggest that G. lucidum has potential to produce laccase enzyme by using substrate comprising corncob and rice straw on submerged fermentation.

Pigment production by Fusarium solani BRM054066 and determination of antioxidant and anti-inflammatory properties

The fungal kingdom has been widely studied as a source of bioactive compounds of interest to the pharmaceutical and food industry. This paper studies the production of natural red pigments by Fusarium solani BRM054066 in the submerged fermentation system, using Doehlert experimental design to determine optimal cultivation conditions. The chemical composition of the red pigment was determined by Nuclear Magnetic Resonance spectroscopy (NMR) and Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Antioxidant activity was assessed by the ability to sequester of free radical DPPH. In the analysis of anti-inflammatory activity, murine peritoneal macrophages activated by LPS were used, and the gene expression of TNF-α, IL-1β, IL-6, IL-10 and IL-17 was determined using qPCR. As a result, it was found that agitation at 200 rpm and glucose concentration ≥ 20 g/L promote the best results in the production of red pigment. The chemical compounds identified were two naphthoquinones, fusarubin and dihydrofusarubin, and an anthraquinone, a bostrycoidin, being fusarubin the majority compound. The red pigment showed antioxidant activity by scavenge 50% of the DPPH radical, in a concentration of 24 µg/mL. The pigment also showed an effective anti-inflammatory capacity by reducing the overexpression of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and promoting the production of anti-inflammatory IL-10 and IL-17, in murine macrophages activated by LPS (p < 0.05). According to the results, the fungus F. solani BRM054066, under optimized conditions of cultivation, proved to be a promising source of biologically active natural pigments with wide industrial applicability.

Effects of Cultural Conditions in Enhancing the Production of Anti-MRSA Activity of Lasiodiplodia pseudotheobromae IBRL OS-64, an Endophytic Fungus Isolated from Leaf of Ocimum sanctum L. in Submerged Fermentation System

Effects of Cultural Conditions in Enhancing the Production of Anti-MRSA Activity of Lasiodiplodia pseudotheobromae IBRL OS-64, an Endophytic Fungus Isolated from Leaf of Ocimum sanctum L. in Submerged Fermentation System

Ellagic acid production using polyphenols from orange peel waste by submerged fermentation

Abstract Background Biotechnological processes are part of modern industry as well as stricter environmental requirements. The need to reduce production costs and pollution demands for alternatives that involve the integral use of agro-industrial waste to produce bioactive compounds. The citrus industry generates large amounts of wastes due to the destruction of the fruits by microorganisms and insects together with the large amounts of orange waste generated during the production of juice and for sale fresh. The aim of this study was used orange wastes rich in polyphenolic compounds can be used as source carbon of Aspergillus fumigatus MUM 1603 to generate high added value compounds, for example, ellagic acid and other molecules of polyphenolic origin through submerged fermentation system. Results The orange peel waste had a high concentration of polyphenols, 28% being condensed, 27% ellagitannins, 25% flavonoids and 20% gallotannins. The major polyphenolic compounds were catechin, EA and quercetin. The conditions, using an experimental design of central compounds, that allow the production of the maximum concentration of EA (18.68 mg/g) were found to be: temperature 30°C, inoculum 2 × 107 (spores/g) and orange peel polyphenols 6.2 (g/L). Conclusion The submerged fermentation process is an effective methodology for the biotransformation of molecules present in orange waste to obtain high value-added as ellagic acid that can be used as powerful antioxidants, antibacterial and other applications. How to cite: Sepulveda L, Laredo-Alcala E, Buenrostro-Figueroa JJ, et al. Ellagic acid production using polyphenols from orange peel waste by submerged fermentation. Electron J Biotechnol 2020;43. https://doi.org/10.1016/j.ejbt.2019.11.002 .

Natural Red Pigment Production by Monascus Purpureus in Submerged Fermentation Systems Using a Food Industry Waste: Brewer's Spent Grain.

This paper studies the production of natural red pigments by Monascus purpureus CMU001 in the submerged fermentation system using a brewery waste hydrolysate, brewer’s spent grain (BSG). The chemical, structural and elemental characterization of the BSG was performed with Van-Soest method, Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS), respectively. The lignocellulosic structure of BSG was hydrolyzed with a dilute sulfuric acid solution (2% (w/v)) followed by detoxification with Ca(OH)2. Maximum red pigment production (22.25 UA500) was achieved with the following conditions: 350 rpm shake speed, 50 mL fermentation volume, initial pH of 6.5, inoculation ratio of 2% (v/v), and monosodium glutamate (MSG) as the most effective nitrogen source. Plackett–Burman design was used to assess the significance of the fermentation medium components, and MSG and ZnSO4·7H2O were found to be the significant medium variables. This study is the first study showing the compatibility of BSG hydrolysate to red pigment production by Monascus purpureus in a submerged fermentation system.

Biological treatment of lignocellulosic biomass by Chaetomium globosporum: Process derivation and improved biogas production.

Chaetomium globosporum was isolated from aeolian soil samples collected from semi-arid locations in the state of Rajasthan, India. The efficiency of fungal strain for biological treatment of biomass to improve biogas yield was screened by estimating laccase enzyme activity under submerged fermentation system. Further, lignocellulosic biomass(s) wheat and pearl millet straw were subjected to biological treatment and subsequent increase in release of reducing sugar as compared to untreated straw was determined. Optimization of biological treatment conditions was accomplished by employing response surface methodology with Box Behnken design of experiments. Impact of three parameters (temperature, residence time, moisture content) was analyzed using three severity levels on output response of biological treatment in terms of released reducing sugar (g/L). Observed values were well fitted with second order polynomial equation (R2 = 0.91) and predicted outcomes were in agreement with experimental results. Optimized treatment conditions were: 36 °C, 31 days residence time and 81% moisture. A 2.9 fold increase in reducing sugar was released when substrates were treated under these conditions. Our research findings emphasize importance of optimizing biological treatment conditions to achieve high biogas yield when using wheat and pearl millet straw as feedstocks and similar treatment could be carried out for other lignocellulosic feedstocks.

Production of Ligninolytic Enzymes by Coptotermes curvignathus Gut Bacteria

Abstract Maximum utilization of lignocellulosic biomass is contingent upon degrading the recalcitrant lignin polymer. Conventional methods employed in delignification require high inputs of energy and chemicals, resulting in the release of highly toxic effluents. The ability of gut flora of Coptotermes curvignathus in lignin degradation was investigated in this study. Production of ligninolytic enzymes was done in an aerated submerged fermentation system with kraft lignin as sole carbon source. The degradation experiment was carried out for 7 days at 30 °C, pH 7. Three potential lignin degraders identified as Bacillus sp., Lysinibacillus sp. and Acinetobacter sp. were successfully isolated. The bacterial growth and secretion of extracellular ligninolytic enzymes confirmed metabolism of kraft lignin by the identified strains. Lysinibacillus sp., a novel lignin degrader showed highest manganese peroxidase (76.36 ± 15.74 U/L) and laccase activity (70.67 ± 16.82 U/L) after 7 and 6 days of incubation respectively, while maximal activity of lignin peroxidase (262.49 ± 0.92 U/L) was recorded after 7 days in culture supernatants of Bacillus sp. With respect to the activity of the secreted enzymes, the lignin degrading potential of these bacterial strains can be explored in the valorisations of lignocellulosic biomass in industrial processes such as pulping, bioethanol production, fine chemicals and materials synthesis.

Comparative study on lipase production using solid state and submerged fermentation systems by several fungal strains and the predicted molecular characteristics

Background: Lipases produced from microbial fermentation systems belong to the group of hydrolase enzymes and have tremendous commercial applications. The performance of solid state and submerged fermentation in producing lipase by several fungal strains was compared and the structure and functions of the lipases were predicted by molecular prediction studies. Methods: Lipase production was investigated using both solid state fermentation (SSF) and submerged liquid fermentation (SLF); with palm kernel cake as the fermentation substrate for SSF. Lipase activity, protease activity, total protein and biomass content were assayed. The predictive molecular characterization techniques were employed to determine possible structure and function of the lipases. Results: All fungal strains tested showed substantially higher lipase activity in SSF compared to SLF except for Trichoderma viride SDTC EDF 002. Aspergillus niger SDTC SRW-4 (44.43 U g-1; 176.36 mg g-1) and Aspergillus niger DSMZ 2466 (42.05 U g-1; 176.28 mg g-1) showed the highest lipase activities and total protein content. All lipases showed the presence of “lipase 3” domain with conserved dyad catalytic site of two residues (Ser-Asp) unlike several other esterase lipases with triad catalytic site (Ser-Asp/Glu-His). Predicted pI range of lipases was 4.22 to 8.28 with molecular weight ranging from 31.6 kDa to 54.1 kDa. Conclusions: Production of lipase by Aspergillus niger SDTC SRW-4 and Aspergillus niger DSMZ 2466 using palm kernel cake as substrate was substantially higher in SSF as compared to SLF. Lipases produced by these fungus were predicted to have similar properties and functionality of triacylglycerol lipases (EC 3.1.1.3).

Optimized production, characterization and application of alkaline proteases from taxonomically assessed microbial isolates from Lonar soda lake, India

Lonar soda lake of India was explored for isolation of the industrially important alkaline protease producers. The isolates from Lonar lake were identified based on their morphological characters, microscopic features, enzymatic profile, biochemical pattern and 16S rRNA partial gene sequences as Brachybacterium sp. LAP214, Bacillus cohnii LAP217, Bacillus pseudofirmus LAP220, Brevibacterium casei LAP223 and Halomonas venusta LAP515 and deposited in Microbial Culture Collection, NCCS, Pune (India) for public access. Production of the alkaline proteases from these isolates was optimized by adopting a one-variable-at-a-time (OVAT) approach in a submerged fermentation system in modified fermentation medium (MFM). More than 3 fold enhancement in the alkaline protease production was recorded from all these isolates after optimization of the physicochemical parameters. Molecular weights of partially purified alkaline proteases from LAP515, LAP214 and LAP223 were recorded as 20.1, 14.3 and 43 kDa respectively. Molecular weights of partially purified alkaline proteases from LAP220 and LAP217 were recorded as 29 kDa. Partially purified alkaline proteases from LAP515, LAP214, LAP220, LAP217 and LAP223 exhibited Vmax values 1000, 1290.323, 526.316, 1250 and 2000 U/mg respectively. Catalytic activities of alkaline proteases from all these isolates were partially and completely inhibited in presence of a serine protease inhibitor PMSF, at the concentrations of 1 and 10 mM respectively. The metal cations Mg2+, Ba2+, and Ca2+ enhanced the catalytic activities of these proteases. Remarkable stability of these alkaline proteases was observed in presence of selected solvents and commercial detergents. These proteases therefore can be used in various biotechnological applications.

Carboxylate groups play a major role in antitumor activity of Ganoderma applanatum polysaccharide

In this paper, the structure difference between the polysaccharides isolated from fruit bodies (FGAP) and submerged fermentation system (SGAP) of Ganoderma applanatum was investigated by means of GPC, HPLC and IR, respectively. And their antitumor activities were evaluated against Sarcoma 180 in vivo. The results showed that FGAP and SGAP were typical polysaccharides with different molecular weights, monosaccharide components, and functional groups. Closely related to the distinct structures, FGAP exhibited a better antitumor activity than SGAP. Moreover, since FGAP contained carboxylate groups rather than SGAP, such groups were chemically introduced into SGAP (CSGAP) by carboxymethylation in order to identify their contribution to antitumor activity. The results demonstrated that the inhibition of CSGAP against Sarcoma 180 in vivo was significantly enhanced by comparison to the native SGAP and even higher than that of FGAP, suggesting that the carboxylate groups played a major role in antitumor activity of G. applanatum polysaccharide.

Wealth from waste: Optimized alkaline protease production from agro-industrial residues by Bacillus alcalophilus LW8 and its biotechnological applications

An efficient alkaline protease producer was isolated from the water of the hyperalkaline-saline Lonar soda lake and identified as Bacillus alcalophilus LW8 by culture-dependent techniques by its morphological, microscopic, biochemical, physiological and molecular characteristics. The 16S rRNA gene sequence was submitted to the GenBank nucleotide repository under accession number KC689353. Alkaline protease production was optimized by adopting a one-variable-at-a-time approach in a submerged fermentation system in modified fermentation medium (MFM). The optimized components of MFM were (w/w) casein (1%), sugarcane molasses (1%), NaCl (1%), ammonium sulphate (0.5%), KH2PO4 (0.05%), K2HPO4 (0.05%) and Na2CO3 (1%). Optimized culture conditions were used for alkaline protease production. The final yield of partially purified alkaline protease after dialysis was 53.35%. The molecular mass of the dialysed alkaline protease was 27kDa. On a Lineweaver–Burk plot, the calculated Km and Vmax values were 24mg/mL and 1000U/mg, respectively. The enzyme was remarkably stable in the pH range 7.0–12.0, with optimum activity at pH 10.0. LW8 alkaline protease was completely inhibited by phenylmethylsulphonyl fluoride at 10mmol/L, indicating that the enzyme belongs to the serine protease class. The metal ions Ca2+, Ba2+, Mg2+, Zn2+, Fe3+, Cu2+ and Mn2+ increased the catalytic activity of partially purified alkaline protease. The protease effectively decomposed the gelatinous coating on an X-ray film, hydrolysed blood clot, a blood-stain from a piece of cotton fabric and hairs from a piece of goat skin.