Production of bioethanol from fermented sugars of sugarcane bagasse produced by lignocellulolytic enzymes of Exiguobacterium sp. VSG-1.

Exiguobacterium sp. VSG-1 was isolated from the soil sample and characterized for the production of lignocellulolytic enzymes. Production of these enzymes by the strain VSG-1 was carried out using steam-exploded sugarcane bagasse (SCB) and found to secrete cellulase, pectinase, mannanase, xylanase, and tannase. The growth and enzyme production were found to be optimum at pH 9.0 and 37 °C. Upon steam explosion of SCB, the cellulose increased by 42 %, whereas hemicelluloses and lignin decreased by 40 and 62 %, respectively. Enzymatic hydrolysis of steam-exploded SCB yielded 640 g/l of total sugars. Fermentation of sugars produced from pretreated SCB was carried out by using Saccharomyces cerevisiae at pH 5.0 and 30 °C. The alcohol produced was calculated and found to be 62.24 g/l corresponding to 78 % of the theoretical yield of ethanol. Hence, the strain VSG-1 has an industrial importance for the production of fermentable sugars for biofuels.

Production of extremely alkaliphilic, halotolerent, detergent, and thermostable mannanase by the free and immobilized cells of Bacillus halodurans PPKS-2. Purification and characterization.

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0-16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.

Salicylic acid and salicylic acid sensitive and insensitive catalases in different genotypes of chickpea against Fusarium oxysporum f. sp. ciceri.

Differential expression of catalase isozymes in different genotypes of chickpea resistant genotypes- A1, JG-315, JG-11, WR-315, R1-315, Vijaya, ICCV-15017, GBS-964, GBM-10, and susceptible genotypes- JG-62, MNK, ICCV-08321, ICCV-08311, KW-104, ICCV-08123, ICC-4951, ICC-11322, ICC-08116 for wilt disease caused by Fusarium oxysporum. f. sp. ciceri (Foc) was analyzed. Salicylic acid (SA) and H2O2 concentrations were determined in control as well as in plants infected with F. ciceri and found that the high and low levels of salicylic acid and H2O2 in resistant and susceptible genotypes of chickpea respectively. Catalase isozyme activities were detected in the gel and found that no induction of new catalases was observed in all the resistant genotypes and their some of the native catalase isozymes were inhibited; whereas, induction of multiple catalase isozymes was observed in all the screened susceptible genotypes and their activities were not inhibited upon Foc or SA treatments. The above results support the possible role of these isozymes as a marker to identify which genotype of chickpea is expressing systemic acquired resistance.

Calcium alginate entrapped preparation of α-galactosidase: its stability and application in hydrolysis of soymilk galactooligosaccharides.

Thermostable α-galactosidase from Aspergillus terreus (GR) was insolubilized using concanavalin A obtained from jack bean extract and in order to maintain the integrity of complex in the presence of its substrate or products, this complex was crosslinked with glutaraldehyde. Soluble α-galactosidase entrapped in calcium alginate retained 82% of enzyme activity whereas, Con A-α-galactosidase complex entrapped in calcium alginate and crosslinked Con A-α-galactosidase complex entrapped calcium alginate retained 74 and 61% activity, respectively. A fluidized bed reactor was constructed for continuous hydrolysis of galactooligosaccharides in soymilk using crosslinked Con A-α-galactosidase complex entrapped calcium alginate. Optimum conditions such as pH (5.0) and temperature (65°C) were the same for all immobilized enzyme preparations and soluble enzyme. Crosslinked Con A-α-galactosidase entrapped complex exhibited enhanced thermostability and showed 62% of activity (38%) after 360 min at 65°C. Entrapped crosslinked Con A-α-galactosidase complex preparation was superior in the continuous hydrolysis of oligosaccharides in soymilk by batch processes compared to the other entrapped preparations. The entrapped crosslinked concanavalin A-α-galactosidase complex retained 95% activity after eight cycles of use.

Purification and characterization of endo-beta-1,4 mannanase from Aspergillus niger gr for application in food processing industry.

A thermostable extracellular beta-mannanase from the culture supernatant of a fungus Aspergillus niger gr was purified to homogeneity. SDS-PAGE of the purified enzyme showed a single protein band of molecular mass 66 kDa. The beta- mannanase exhibited optimum catalytic activity at pH 5.5 and 55 degrees C. It was thermostable at 55 degrees C, and retained 50% activity after 6 h at 55 degrees C. The enzyme was stable at a pH range of 3.0 to 7.0. The metal ions Hg(2+), Cu(2+), and Ag(2+) inhibited complete enzyme activity. The inhibitors tested, EDTA, PMSF, and 1,10-phenanthroline, did not inhibit the enzyme activity. N-Bromosuccinimide completely inhibited enzyme activity. The relative substrate specificity of enzyme towards the various mannans is in the order of locust bean gum>guar gum>copra mannan, with K(m) of 0.11, 0.28, and 0.33 mg/ml, respectively. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food-processing industry.

Three-phase partitioning of alpha-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques.

Simple, attractive and versatile technique, three-phase partitioning (TPP) was used to purify alpha-galactosidase from fermented media of Aspergillus oryzae. The various conditions required for attaining efficient purification of the alpha-galactosidase fractions were optimized. The addition of n-butanol, t-butanol, and isopropanol in the presence of ammonium sulfate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The single step of three-phase partitioning, by saturating final concentration of ammonium sulfate (60%) with 1:1 t-butanol, gave activity recovery of 92% with 12-fold purification at second phase of TPP. The final purified enzyme after TPP showed considerable purification on SDS-PAGE with a molecular weight of 64 kDa. The enzyme after TPP showed improved activity in organic solvents. Results are compared with conventional established processes for the purification of alpha-galactosidase produced by Aspergillus oryzae and overall the proposed TPP technique resulted in 70% reduction of purification cost compared to conventional chromatographic protocols.

Purification and characterization of thermostable alpha-galactosidase from Aspergillus terreus (GR).

An extracellular thermostable alpha-galactosidase producing Aspergillus terreus (GR) strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 degrees C, respectively, for artificial substrate pNPalphaGal. alpha-Galactosidase from A. terreus (GR) is found to be thermostable, as it was not inactivated after heating at 65 degrees C for 40 min. The K (m) for pNPalphaGal, oNPalphaGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg(2+), Ni(2+), Ca(2+), Co(2+), and Mn(2+) had no effect on enzyme activity, but Ag(+), Hg(2+), and Cu(2+) have inhibited complete activity.

Optimization of the production of thermostable endo-beta-1,4 mannanases from a newly isolated Aspergillus niger gr and Aspergillus flavus gr.

The aim of this work was to establish optimal conditions for the maximum production of endo-beta-1,4 mannanases using cheaper sources. Eight thermotolerant fungal strains were isolated from garden soil and compost samples collected in and around the Gulbarga University campus, India. Two strains were selected based on their ability to produce considerable endo-beta-1,4 mannanases activity while growing in liquid medium at 37 degrees C with locust bean gum (LBG) as the only carbon source. They were identified as Aspergillus niger gr and Aspergillus flavus gr. The experiment to evaluate the effect of different carbon sources, nitrogen sources, temperatures and initial pH of the medium on maximal enzyme production was studied. Enzyme productivity was influenced by the type of polysaccharide used as the carbon source. Copra meal defatted with n-hexane showed to be a better substrate than LBG and guar gum for endo-beta-1,4 mannanases production by A. niger gr (40.011 U/ml), but for A. flavus gr (33.532 U/ml), the difference was not significant. Endo-beta-1,4 mannanases produced from A. niger gr and A. flavus gr have high optimum temperature (65 and 60 degrees C) and good thermostability in the absence of any stabilizers (maintaining 50% of residual activity for 8 and 6 h, respectively, at 60 degrees C) and are stable over in a wide pH range. These new strains offer an attractive alternative source of enzymes for the food and feed processing industries.

Purification of alpha-galactosidase and invertase by three-phase partitioning from crude extract of Aspergillus oryzae.

Alpha-galactosidase and invertase were accumulated in a coherent middle phase in a three-phase partitioning system under different conditions (ammonium sulphate, ratio of tert-butanol to crude extract, temperature and pH). Alpha-galactosidase and invertase were purified 15- and 12-fold with 50 and 54% activity recovery, respectively. The fractions of interfacial precipitate arising from the three-phase partitioning were analyzed by SDS-PAGE. Both purified preparations showed electrophoretic homogeneity on SDS-PAGE.

Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation.

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.

Isolation and identification of alpha-amylase producing Bacillus sp. from dhal industry waste.

A bacterial strain was isolated from dhal industry red gram waste and identified as Bacillus. A thermostable extracellular amylase was partially purified from the strain. Optimum temperature and pH for the enzyme were found to be 60 degrees C and 6.5, respectively. The maximum amylase production was achieved with maltose as carbon source. Among the nitrogen sources, peptone and yeast extract produced maximum amylase.

Oligosaccharins of black gram (Vigna mungo L.) as affected by processing methods.

The oligosaccharide content was determined in 12 different cultivars of black gram. The effect of various treatments such as soaking, cooking, and enzyme treatment on the raffinose family oligosaccharides of dry seeds and flour was studied. Ajugose, a higher oligosaccharide (DP 6) found in trace quantities in seeds, was shown in black gram by HPLC. The percent reduction of raffinose, stachyose, verbascose, and ajugose after soaking for 16 hr was 41.66%, 47.61%, 28.48%, and 26.82%, respectively in Local-I variety and 43.75%, 20.58%, 23.60%, and 15.88%, respectively in Local-II variety. Cooking for 60 min resulted in decrease of 100% for raffinose, 76.19% for stachyose, 36.39% for verbascose, and 60.97% for ajugose in Local-I variety and 100% for raffinose, 55.88% for stachyose, 48.52% for verbascose, and 56.07% for ajugose in Local-II variety. Thin layer chromatographic analysis of 3 hr enzyme-treated samples revealed almost complete hydrolysis of raffinose family of oligosaccharides. Among the different methods employed, enzyme treatment was found to be the most effective for removing alpha-galactosides in black gram.

Stability and activity of potato acid phosphatase in aqueous surfactant media.

The effect of three different classes of surfactants viz., anionic, cationic and neutral on catalytic activity of potato acid phosphatase (AcPase) was studied. Anionic surfactants bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and sodium dodecyl sulfate (SDS) inhibited AcPase activity completely at 3 mM concentration. The cationic surfactant cetyltrimethylammonium bromide (CTAB), at 3.33 mM enhanced the activity by 20 per cent. Increase in CTAB concentration decreased the activity, so that only 50 per cent was retained at 27 mM concentration. Brij 35, a neutral surfactant also decreased the activity, whereas TritonX-100 had little or no effect. At low CTAB concentration, an increase in Vmax and a decrease in Km was observed and the V/K ratio increased. TritonX-100 enhanced Vmax without changing Km. The V/K ratio was also increased. The UV-difference spectra of AcPase in presence of CTAB showed perturbations. The intensity of intrinsic fluorescence also enhanced in presence of CTAB.

Superactivity and phase-sensitivity of potato acid phosphatase entrapped in reverse micelles.

Potato acid phosphatase, AcPase (E.C. 3.1.3.2) was entrapped in reverse micelles of cationic surfactant cetyltrimethylammonium bromide (CTAB) in isooctane and chloroform (1:1). The activity was studied at different values of Wo = ([water]/[surfactant]). AcPase exhibited superactivity in the reverse micellar system. At very low Wo value, activity was found to be less than that of in buffer and further increase in Wo value enhanced the activity thousand fold. At Wo = 50, the activity was enhanced more than twenty fold. The effect of second surfactant, TritonX-100, on superactivity was studied. There was a slight decrease in overall activity, when 3.33 mM TritonX-100 was added to the above reverse micellar system.

Enzymic hydrolysis of raffinose and stachyose in soymilk by alpha-galactosidase from Gibberella fujikuroi.

The use of intracellular alpha-galactosidase from Gibberella fujikuroi to remove raffinose and stachyose in soymilk was studied. The optimum conditions for the enzymic hydrolysis of raffinose and stachyose was pH 5.5 to 6.0 at 55 degrees C. Alpha-galactosidase showed optimum activity at pH 5.0 and 50 degrees C with the substrate p-nitrophenyl-alpha-D-galacto-pyranoside (PNGP). The enzyme showed no detectable loss of activity when held more than 8 hr at 50 degrees C. Thin layer chromatography (TLC) revealed the following composition of oligosaccharides in local soybean variety: sucrose, 5.53%; raffinose, 1.95%; and stachyose, 6.1%. Investigation by TLC showed complete hydrolysis of raffinose and stachyose in 3 hr. HPLC analysis of hydrolyzate indicated complete hydrolysis of stachyose, and more than 60% hydrolysis of raffinose in 2.5 hr.

Changes in trypsin and chymotrypsin inhibitory activity on soaking of redgram (Cajanus cajan L.).

Changes in trypsin and chymotrypsin inhibitory activity of redgram seeds on soaking in distilled water, different salt solutions and mixed salt solution were investigated. Reduction in trypsin and chymotrypsin inhibitory activity was observed on soaking in salt solutions in comparison to soaking in distilled water. Maximum loss of trypsin and chymotrypsin inhibitory activity was observed on soaking the seeds in mixed salt solution.

Tannic acid content in sorghum (Sorghum bicolour M.): effects of processing.

Some simple treatments were employed to reduce the tannin content in locally consumed sorghum grain. The treatments included overnight soaking of sorghum in 2% NaHCO3, soaking in different alkalis, ammoniation and autoclaving. Of the above treatments, ammoniation was best for complete removal of tannins. Soaking the seeds in alkalis was also effective. Soaking the sorghum seeds for 18 hours in mixed salt solution (containing 1.5% NaHCO3 + 0.5% Na2CO3 and 0.75% citric acid in w/v ratio) was also found to be effective.

Effects of infrared radiation, solar cooking and microwave cooking on alpha-amylase inhibitor in sorghum (Sorghum bicolor L.).

Three domestic cooking methods were studied in alpha-amylase inhibitory activity in sorghum grains. In all the treatments, overnight soaked seeds lost amylase inhibitory activity much faster. All the three treatments reduced the inhibitory activity. Use of solar cooker for reducing amylase inhibitory activity works out very economically and efficiently. Microwave cooking eliminates amylase inhibitory activity within 5 minutes.

Effect of heat treatments on trypsin/chyomotrypsin inhibitor activity of red gram (Cajanus cajan L.).

The application of dry heat to the seeds and meal was not effective in inactivating the trypsin inhibitory activity (TIA) and chymotrypsin inhibitory activity (CIA). Soaking for 24 hours followed by cooking for 20 min was effective in destroying the TIA and CIA.

Effect of heat treatment and germination on alpha amylase inhibitor activity in chick peas (Cicer arietinum L.).

Chick pea seeds of twenty eight varieties were analysed for alpha amylase inhibitor activity (AIA) using salivary amylase. The effects of heat treatment and germination on the activity of the antinutritional factor was investigated. Heat treatment and germination decreased the activity of amylase inhibitor. Chick pea meal was also subjected to UV irradiation and pressure cooking. These treatments decreased alpha amylase inhibitor activity. The amylase inhibitor activity decreased as the days of germination increased and negligible inhibitor activity was observed on the 6th day of germination.