Optimization and Characterization of Thermostable Endo and Exocellulases by <i>Humicola sp</i>. SKESMBKU03

The biomass yield, cellulolytic activity, and protein recovery using Aspergillus terreus GN1 with alkali-treated sugarcane bagasse was studied using different levels (250-600 mg of N/L of broth) of organic and inorganic nitrogen sources. e.g., cattle urine, urea, cornsteep liquor, ammonium sulfate, ammonium nitrate, ammonium iron sulfate, ammonium chloride, and sodium nitrate. Among different levels of alkali-treated bagasse substrate concentrations (0.5-4.0% w/v) tested, 1.0% substrate yielded the highest crude protein content, protein recovery, and cellulolytic activity. The biomass recovery with 1.0% substrate ranged from 290-380 mg/500 mg bagasse substrate in a 50-mL broth with a nitrogen level of 250-600 mg of N/L in all the sources except ammonium iron sulfate, which yielded 402-439 mg/500 mg bagasse substrate. However, crude protein content of biomass obtained with an ammonium iron sulfate nitrogen source was the lowest. Cornsteep liquor nitrogen source at the rate of 600 mg of N/L yielded the maximum crude protein of 32.9%, protein recovery of 22.2 g/100 g of bagasse, and carboxymethyl cellulase and filter paper enzyme activities of 1.1 and 0.09 units/mL, among the organic and inorganic nitrogen sources studied. In general, the organic nitrogen sources and inorganic nonammonium nitrogen sources were utilized preferentially for protein production over the inorganic ammonium nitrogen sources. The fermentation time required under optimum cultural and nutritional conditions for A. terreus GN1 was also evaluated. The crude protein content of the biomass increased gradually up to the seventh day of fermentation, but the protein recovery rate was high up to two or three days. It was observed that the cellulose utilization rate increased after an initial lag of one day up to the third day and gradually increased further, which corresponded positively with protein content, biomass protein recovery, and cellulase enzyme activity. On the seventh day of fermentation, the crude protein content, biomass protein recovery, water-soluble carbohydrate, bagasse cellulose utilization, CMCase, and FPase activities were 32.8%, 20.1 g/100 g of bagasse, 6.2%, 82.7%, 1.0. and 0.08 U/mL, respectively. The final biomass recovered contained 32.8% crude protein content and had an in vitro rumen digestibility (IVRD) coefficient of 68.8%. The biomass contained almost all the essential and nonessential amino acids and was comparable with FAO reference protein. It is concluded that a fermentation time of 72 h gave a faster rate of protein production of 16.9 g/100 g of bagasse with 69.8% bagasse cellulose utilization with 76.0% IVRD. and contained almost all the essential and nonessential amino acids.

L-glutaminase has attracted much attention due its wide range of applications in several fields. The L-glutaminase widely used in pharmaceutical and food industries. L-glutaminase is generally regarded as a key enzyme that controls the delicious taste of fermented foods such as soy sauce. L-glutaminase production was carried out by using supplementation of organic and inorganic nitrogen sources such as yeast extract, malt extract, peptone and urea at concentration ranging from 0.25% to 1.25% with increments of 0.25% and also different inorganic nitrogen sources like ammonium sulphate and ammonium chloride at concentration ranging from 0.025% to 0.125% with increments of 0.025%. The malt extract (1%) produced 399.9 IU, were best organic nitrogen source and ammonium sulphate (0.1%) appear to be good inorganic nitrogen source under submerged fermentation process and showed 546 IU. Current study is an exploring step to industrial sector to upscale their L-glutaminase production and it will useful strategy to commercial sector and alternative to old methods

Some fungi in the Chytridiomycota can assimilate both inorganic and organic sources of nitrogen

The current study is designed having aims i.e., to check cellulase production potential of P. spadiceus, optimization of different culture conditions for the production of exoglucanase using sugarcane bagasse as substrate and characterization of exoglucanase. P. spadiceus, a mushroom was used to enhance the production of exoglucanase enzyme by Solid State Fermentation (SSF). Effect of different conditions was observed including, fermentation period, temperature, pH, moisture level and different carbon and nitrogen sources to obtain maximum yield of exoglucanase. Crude-enzyme was then centrifuged and characterized for temperature, pH, effect of metal ions and effect of different substrate concentration for the determination of Km and Vmax. Maximum activity of exoglucanase was observed after day 4 (57.64 IU/mL/min), at 25-30oC and 50% moisture level (31.5 IU/mL/min), at pH 4.5 (47.03 IU/mL/min) and also with carbon source (5% sucrose) and nitrogen source (2.5% urea) which was 39.2 IU/mL/min. The enzyme was subjected to characterization to find out its optimum temperature, pH, effect of different metal ions and finding Km and Vmax. Exoglucanase activity at different conditions of each parameter showed that the optimum temperature was 30oC and pH 4.5. The Km and Vmax were determined by the Line-weaver-Burk-plot between 1/[VO] on the Y-axis and 1/[S] on the X-axis that results 66.66 mM and 48.73 µM/mL/min respectively The study of exoglucanase enzyme production from this fungus and to optimize different conditions will aid in more enzyme production, decrease environmental pollution and produce valued things from the resulting fermentable-sugars.

Inulin fructotransferase is an enzyme which converts inulin into difructose anhydride III (DFA III). Inulin fructrotransferase was produced by fermentation in medium containing various concentrations of inulin (10 g/L, 20 g/L) as carbon source, yeast extract (1 g/L, 2g/L) as organic nitrogen source and (NH4)2SO4 (2g/L, 5g/L) as inorganic nitrogen source using Nonomuraea sp. ID06-A0189. The aim of the study was to optimize the medium composition using various concentrations of carbon and nitrogen sources for inulin fructotransferase production by Nonomuraea sp. ID06-A0189, through shake flask system. Fermentation was conducted at 30 °C, pH 7.0 and agitated in 130 rpm for three days. The result showed that comparable with those found in another condition the highest enzyme activity was produced in medium using inulin as carbon source with concentration of 10 g/L, yeast extract as organic nitrogen source with concentration of 5 g/L and ammonium sulfate as inorganic nitrogen source with concentration of 2 g/L. Addition of inulin as a carbon source increased the volume of biomass by about 11.5-24 mL. The enzyme activity in the culture supernatant reached 25.3 U/mL after three days incubation.

The main aim of the study is to enhance the biosynthesis of xylanse by incorporating nitrogen sources (both organic and inorganic). Recently, xylanases have expanded their use in many processing industries, such as pulp and paper, food and textile to newer needs such as biofuel production. .This study were taken up to the enhance the biosynthesis of xylanase by supplementation of organic nitrogen and inorganic nitrogen sources were employed in range of 0.25% to 1.25%. The organic nitrogen source were supplemented are peptone, yeast extract and beef extract and inorganic nitrogen sources are ammonium sulphate, ammonium chloride and ammonium nitrate. The yeast extract and ammonium nitrate were yielded higher xylnase production and showed 5.56 IU and 5.79 IU Key words: Xylanase, submerged fermentation, yeast extract, ammonium nitrate and assay.

The effect of organic and inorganic nitrogen sources on Trichoderma reesei Rut-C30 cellulase production was investigated in submerged cultivations. Stirred tank bioreactors and shake flasks, with and without pH control, respectively, were employed. The experimental design involved the addition of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml(-1)) were obtained with media containing soy peptone (3-6 g l(-1)) and glutamate (3.6 g l(-1)). However, these improvements in the cellulase titers in the presence of the organic nitrogen sources appeared to be related to smaller changes in the pH of the medium. This was confirmed using stirred tank bioreactors with pH control. No significant differences were observed in the highest cellulase titers and the protein pattern (according to the SDS-PAGE) of supernatants from pH controlled stirred tank bioreactor cultivations, when different nitrogen sources were used in the medium. Here the cellulase activities (~1.0 ± 0.2 FPU ml(-1)) were also much greater (8-150 times) than in shake flask cultivation. Consequently, the addition of ammonium sulphate as sole nitrogen source to Avicel basal medium is recommended when performing cultivations in stirred tank bioreactors with strict pH controlled conditions.

Recently, xylanases have expanded their use in many processing industries, such as pulp and paper, food and textile to newer needs such as biofuel production. .This study were taken up to the enhance the biosynthesis of xylanase by supplementation of organic nitrogen and inorganic nitrogen sources were employed in range of 0.25% to 1.0%. The organic nitrogen source were supplemented are peptone, yeast extract and beef extract and inorganic nitrogen sources are ammonium Sulphate and ammonium chloride. The beef extract and ammonium nitrate were yielded higher xylnase production and showed 7.5 IU and 8.46 IU Key words: Xylanase, submerged fermentation, xylose, fermentation kinetics and inoculums size

Sclerotial blight of tea (Camellia sinensis L.) caused by Sclerotium rolfsii Sacc. is one of the destructive diseases in tea growing areas of the world. In the present investigation, an attempt was made to know the optimum conditions for the mycelial growth of S. rolfsii. Factors influencing mycelial growth of S. rolfsii were studied with special reference to their growth in different media, variable pH and variable sources of carbon (viz., 6 types) as well as organic (viz., 4 types) and inorganic (viz., 4 types) nitrogen sources. Maximum growth of pathogen occurred after 8 days of inoculation at pH 6. Dextrose was the most effective carbon source and yeast extract (organic source) was found most optimum for growth of S. rolfsii. Organic nitrogen sources were found to be better than inorganic nitrogen sources. DOI: http://dx.doi.org/10.3126/njbs.v1i0.7466 Nepalese Journal of Biosciences 1: 26-31 (2011)

Heavy-ion beams, possessing a wide mutation spectrum and increased mutation frequency, have been used effectively as a breeding method. In this study, the heavy-ion beams generated by the Heavy-Ion Research Facility in Lanzhou were used to mutagenize Aspergillus terreus CA99 for screening high-yield lovastatin strains. Furthermore, the main growth conditions as well as the influences of carbon and nitrogen sources on the growth and the lovastatin production of the mutant and the original strains were investigated comparatively. The spores of A. terreus CA99 were irradiated by 15, 20, 25, and 30 Gy of 80 MeV/u (12)C(6+) heavy-ion beams. Based on the lovastatin contents in the fermentation broth, a strain designated as A. terreus Z15-7 has been selected from the clone irradiated by the heavy-ion beam. When compared with the original strain, the content of lovastatin in the fermentation broth of A. terreus Z15-7 increased 4-fold. Moreover, A. terreus Z15-7 efficiently used the carbon and nitrogen sources for the growth and production of lovastatin when compared to the original strain. The maximum yield of lovastatin, 916.7 μg/ml, was obtained as A. terreus Z15-7 was submerged cultured in the chemically defined medium supplemented with 3% glycerol as a carbon source, 1% corn meal as an organic nitrogen source, and 0.2% sodium nitrate as an inorganic nitrogen source at 30 °C in the shake flask. The result shows that heavy-ion beam irradiation is an effective method for the mutation breeding of lovastatin production of A. terreus.

The study was conducted in order to create the best conditions (physical and chemical factors) that necessary for the growth of the Bacillus subtilis and its production of the Xylanase enzyme. The results include isolation and diagnosis of (40) isolates of bacteria: Bacillus subtilis obtained from (30) soil samples and study the effect of some physical factors (period and temperature of incubation, pH of the medium, ventilation) on bacterial growth and its production of an Xylanase enzyme. The results showed that best growth and its production of the enzyme was obtained when the incubation is (48) hours and the temperature (30) Co , pH of the medium (7) and incubation with shaking at (200) rpm where the optical density value of bacterial growth was reached (0.25) and activity of the enzyme product was (3.41) unit / ml. The effect of some chemical agents (carbon and nitrogen source for medium) on bacterial growth and its production of an Xylanase enzyme and the results revealed that the carbon source (Xylan) gave the best growth and production of the enzyme compared with other of carbon sources (Starch, Cellulose, Xylose, Glucose, Sucrose, Maltose, Lactose), where the optical density value of growth was (0.25) and the enzyme activity (3.41) unit / ml. Regarding the effect of the nitrogen source (organic and inorganic) in medium on growth and production of the enzyme, the results showed that the best bacterial growth and the production of an Xylanase enzyme occur when organic nitrogen sources (Peptone, Beef extract, Yeast extract, Casien) were supplemented in the medium compared with inorganic nitrogen sources (KNO3, (NH4) 2SO4, NH4NO3, NaNO3). Peptone and Yeast extract were the best nitrogen sources for stimulation of bacterial growth and the production of the enzyme with optical density value of growth reached (0.29 , 0.286) and the enzyme activity was (3.92 , 3.9) unit / ml.

This study examined the optimization of growth conditions of Serratia marcescens isolated from a loamy soil of the Federal University Technology Minna Nigeria, for prodigiosin production. The different optimized conditions examined were extraction solvents, incubation time, pH, temperature, carbon sources, organic nitrogen sources, inorganic nitrogen sources and agitation levels. The result reveals that methanol is an ideal solvent recording absorbance of 0.69nm, which is slightly followed by ethanol and acetone with absorbance of 0.4nm and 0.3nm respectively. The bacterium produced maximum level of pigment after 36 hours of incubation (22.20µg/L), although pigment production was observed from 12 hours of incubation onwards (7.40µg/L). The pigment production declined after 36 hours of incubation. The results also revealed that pigment production by S. marcescens was maximum at 25°C (25.10µg/L) followed by 30oC (22.50µg/L). The optimal production was obtained at pH 7 (25.00mg/ml) with dextrose as optimal source of carbon (22.40mg/ml). There was no pigment production at static condition but maximum pigment production was recorded at 150 rpm (22.50µg/L). All the inorganic nitrogen sources caused a reduced biomass production. Among the organic nitrogen sources tested, yeast extract supported maximum pigment (26.75µg/L) but peptone led to a decreased pigment production (9.15µg/L) compared to other organic sources. Addition of urea did not support pigment as well as biomass production. Statistical analysis shows significant differences (p<0.05) in prodigiosin productions with different substrates, temperature and agitation levels. The study revealed that the production of prodigiosin was significantly influenced by the extraction solvents, incubation time, pH, temperature, carbon sources, organic nitrogen sources, inorganic nitrogen sources and agitation levels.

The heterologous production of iso-migrastatin (iso-MGS) was successfully demonstrated in an engineered S. lividans SB11002 strain, which was derived from S. lividans K4-114, following introduction of pBS11001, which harbored the entire mgs biosynthetic gene cluster. However, under similar fermentation conditions, the iso-MGS titer in the engineered strain was significantly lower than that in the native producer - Streptomyces platensis NRRL 18993. To circumvent the problem of low iso-MGS titers and to expand the utility of this heterologous system for iso-MGS biosynthesis and engineering, systematic optimization of the fermentation medium was carried out. The effects of major components in the cultivation medium, including carbon, organic and inorganic nitrogen sources, were investigated using a single factor optimization method. As a result, sucrose and yeast extract were determined to be the best carbon and organic nitrogen sources, resulting in optimized iso-MGS production. Conversely, all other inorganic nitrogen sources evaluated produced various levels of inhibition of iso-MGS production. The final optimized R2YE production medium produced iso-MGS with a titer of 86.5 mg/L, about 3.6-fold higher than that in the original R2YE medium, and 1.5 fold higher than that found within the native S. platensis NRRL 18993 producer.

Macrocybe gigantea (Massee) Pegler & Lodge collected from the wild source is a known edible species of mushroom. Thus, for the domestication of this wild mushroom, an attempt has been made to evaluate the best carbon and nitrogen sources supporting its vegetative growth. From the 15 carbon sources evaluated, glucose gave best vegetative growth (9.1mg mL ) amongst the monosaccharides followed by galactose -1 mannose, fructose, ribose and arabinose, whereas the organic acids as carbon sources did not show encouraging results. Amongst the nitrogen sources tested, the complex organic nitrogen source in the form of yeast extract gave the best mycelial growth (7.79 mg mL ) followed by peptone (7.58 mg mL ) whereas -1 -1 inorganic nitrogen source in the form of sodium nitrate (NaNO ), and simple organic nitrogen sources such 3 as acrylamide, niacinamide and urea did not give any growth.. KEYWORDS :Domestication, dry weight, edible, edible mycelium

A halotolerant and highly effective phenol-degrading strain, named as JDD1H, was isolated from soil contaminated by petroleum and identified as Rhodococcus sp. Phenol degradation by strain JDD1H in the presence of high salinity, especially the effect of exotic stimulants such as carbon source, nitrogen source and surfactant on phenol degradation was investigated. The results show that strain JDD1H could utilize phenol as sole carbon and energy source to grow in the presence of 5% NaCl with 16 h adaptive phase. The degradation efficiency of 150 mg/L phenol was up to 99.5% within 40 h. Lactose, cane sugar, glucose and mannitol in wide range of 100~2000 mg/L enhanced phenol degradation significantly. Organic nitrogen sources, as peptone and yeast extract, and inorganic nitrogen sources, as NH4Cl and (NH4)2SO4, accelerated phenol degradation. Tween-80 with concentration of 50-1000 mg/L restrained phenol degradation and Tween-80 of 1500-2000 mg/L could promote degradation.