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Epirubicin is an antineoplastic agent of anthracycline antibiotic, used for treating a variety of tumor types such as lymphoma, cancer of the breast, lung, ovary and stomach. The objective of this work was to demonstrate direct radiolabeling of epirubicin with 99m Tc, quality control, biological characterization and scientigraphic evaluation in tumor bearing mice. The 99mTc-epirubicin labeling was optimized by varying the amounts of ligand 100-350μg, stannous chloride dihydride 20-50μg and pH range 2-10 by using NaOH or HCl. The radiochemical purity of 99m Tc-epirubicin was evaluated by chromatographic techniques (Whatman No. 3 paper and ITLC-SG). HPLC analyses were performed to check purity of epirubicin and radiochemical purity of labeled 99m Tc- epirubicin. Biodistribution and scintigraphic imaging of 99m Tc-epirubicin was performed in normal and tumor bearing mice at various time intervals. The optimum conditions ensuring 99m Tc-epirubicin labeling yield as high as 99% by adding 35μg SnCl 2 .2H 2 O, 200μg of ligand at pH 6 for 30 min at room temperature (25°C±2°C). HPLC of 99m Tc-epirubicin shows about 99% binding of the compound with technetium-99m. Electrophoresis study indicated the neutral nature of 99m Tc-epirubicin. Biodistribution data and scintigraphic results showed that 99m Tc-epirubicin accumulated in the liver as well as in tumor with significant uptake and excellent retention. 99m Tc-epirubicin shows good stability in human serum. In vitro and in vivo studies revealed the significantly uptake of 99m Tc-epirubicin in the tumor, and also indicating the efficiency of 99m Tc-epirubicin as a tumor diagnostic agent.

Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this manuscript.

Commercial scale degradation of hemicelluloses into easily accessible sugar residues is practically crucial in industrial as well as biochemical processes. Xylanolytic enzymes have a great number of possible applications in many biotechnological processes and therefore, these enzymes are continuously attracting the attention of scientists. Due to this fact, different β-Xylosidases have been isolated, purified and characterized from several bacteria and fungi. Microorganisms in this respect have gained much momentum for production of these significant biocatalysts with remarkable features. It is difficult to propagate microorganisms for efficient and cost-competitive production of β-Xylosidase from hemicelluloses due to expensive conditions of fermentation. The screening of new organisms with an enhanced production of β-Xylosidases has been made possible with the help of recombinant DNA technology. β-Xylosidase genes haven been cloned and expressed on large scale in both homologous and heterologous hosts with the advent of genetic engineering. Therefore, we have reviewed the literature regarding cloning of β-Xylosidase genes into various hosts for their heterologous production along with sequence similarities among different β-Xylosidases. The study provides insight into the current status of cloning, expression and sequence analysis of β-Xylosidases for industrial applications.

Amounts of DNA damage and homocysteine (Hcy) in heart patients blood may have strong function in the causation of cardiovascular disease (CVD). The main objective of this work was to know experimentally the role of total oxidants (produced by Reactive Oxygen species (ROS), clinical biochemical indices, their oxidized products and total antioxidant status (TAS) among such patients to find the association of homocysteine, total oxidation status (TOS) and oxidative DNA damage with other clinical parameters in sixty positive CVD patients compared with those of 60 normal subjects. As compared to healthy individuals, CVD patients had significantly higher concentrations of homocysteine (p<0.0001), total oxidants stress (TOS) (p<0.0001), serum total lipids (p<0.04), malondialdehyde (MDA) (p<0.001), high density lipoprotein-cholesterol (HDL-C) (p<0.0001), and low density lipoprotein cholesterol (LDL-C) (p<0.01), than those of healthy individuals. Plasma Hcy content, TOS and amount of DNA were positively and significantly associated with cholesterol, triglycerides, systolic blood pressure, urea, and albumin (p values<0.01). TOS, Hcy and oxidative DNA damage were negatively correlated with HDL-c, TAS and proteins. It is suggested that these parameters have pivotal role in diagnostic process of determining severity in CAD patients. Oxidized products of macromolecules in blood of CVD patients impart major functions in causing CVD disease.

Thermostable enzymes derived from Thermotoga maritima have attracted worldwide interest for their potential industrial applications. Structural analysis and docking studies were preformed on T. maritima β-glucosidase enzyme with cellobiose and pNP-linked substrates. The 3D structure of the thermostable β-glucosidase was downloaded from the Protein Data Bank database. Substrates were downloaded from the PubCehm database and were minimized using MOE software. Docking of BglA and substrates was carried out using MOE software. After analyzing docked enzyme/substrate complexes, it was found that Glu residues were mainly involved in the reaction, and other important residues such as Asn, Ser, Tyr, Trp, and His were involved in hydrogen bonding with pNP-linked substrates. By determining the substrate recognition pattern, a more suitable β-glucosidase enzyme could be developed, enhancing its industrial potential.

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to β-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

Bacterial infection is found to be the cause of death throughout the world. Nuclear medicine imaging with the help of radiopharmaceuticals has great potential for treating infections. In the present work, clindamycin, a lincosamide antibiotic, was labeled with technetium-99 m (~380 MBq). Clindamycin has been proven to be efficient for treating serious infections caused by bacteria such as Staphylococcus aureus. Quality control, characterization, biodistribution, and scintigraphy of radiolabeled clindamycin were done, and labeling efficiency was determined by ascending paper chromatography. More than 95 % labeling efficiency with technetium-99 m ((99m)Tc) was achieved at pH 6-7 while using 2.5-3 μg SnCl2 · H2O as a reducing agent and 100 μg of ligand at room temperature. The characterization of the compound was performed by using electrophoresis, HPLC and shake flask assay. Electrophoresis indicates the neutral behavior of (99m)Tc-clindamycin. HPLC analysis confirms the single specie of the labeled compound, while shake flask assay confirms high lipophilicity. The biodistribution studies of (99m)Tc-clindamycin were performed Sprague Dawley rats bearing bacterial infection. Scintigraphy and biodistribution studies showed a high uptake of (99m)Tc-clindamycin in the liver, heart, lung, and stomach as well as at S. aureus-infected sites in rabbits.

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48 ± 0.02 g g(-1)), theoretical yield (93 ± 3%), and extracellular invertase productivity (1,430 ± 50 IU l(-1) h(-1)), respectively, when fermenting 180 g sugars l(-1) in molasses medium at 43 °C in 300 m(3) working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M(-1)) and entropy (ΔS*) (-202.88 J M(-1) K(-1)) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l(-1) h(-1)) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.

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 Q p , Y p/s , Y p/X , and q p were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112 kDa. The apparent K m and V max 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.

Enzymes from thermophilic organisms are believed to be strong candidates for industrial applications due to their ability to withstand temperature-induced enzyme inactivation. The present study demonstrated molecular cloning, over-expression, purification and characterization of β-glucosidase from Thermotoga maritima. The bglA gene with a capacity to encode a 51 kDa enzyme was heterologously expressed in E. coli M15. The enzyme was produced @130 mgL(-1) in LB media and @440 mgL(-1) in Dubos salt medium accounting 40-47 % of total cellular soluble proteins when lactose was used as an inducer. The enzyme showed a peak activity between pH and temperature range of 5.0-7.0 and 80-100 °C, respectively. The activity was fairly stable up to 140 °C. The turnover rate (kcat) of the enzyme was 187.1±20 s(-1), whereas the Km and Vmax values were 0.56 mM and 238±2.4 IU mg(-1) protein, respectively. The enzyme was shown to have half-life of 136, 71 and 12.6 h at 80, 90 and 100 °C, respectively. Thermodynamics parameters including melting temperature (130 °C), activation energy for inactivation (36.92 kJmole(-1)), enthalpy (33.73 kJmole(-1)), Gibb's free energy (127.96 kJmole(-1)) and entropy (-246.46 Jmole(-1)K(-1)) have shown that the enzyme have enhanced hydrophobic interactions to prevent its thermal unfolding. These features endorse the industrial applications of the enzyme.

Detailed kinetic and thermodynamic parameters for lysine production from Brevibacterium lactofermentum are investigated for the first time in this study. Production of the essential amino acid, L-lysine, by B. lactofermentum was assessed in a flask and a continuously stirred tank fermentor (22 L). Maximum lysine production was achieved after 40 h of growth and at 35 °C. The effect of different nitrogen sources such as NH(4)NO(3), (NH(4))(2)SO(4), (NH(4))(2)HPO(4), corn steep liquor, NaNO(3), and urea showed that corn steep liquor gave a better lysine yield. Lysine production was increased when dissolved oxygen was maintained at 50 % saturation. The use of dissolved oxygen was critical for high productivity. This indicates that dissolved oxygen greatly affects L-lysine productivity. Kinetic and thermodynamic parameters during lysine production from molasses and glucose mixture showed that B. lactofermentum efficiently converted the substrate mixture into cell mass and lysine. Kinetic and thermodynamic parameters were significantly higher compared with other microorganisms which may be due to the high metabolic activity of B. lactofermentum. This study will have a significant impact on future strategies for lysine production at industrial scale.

Polyhexamethylene biguanide (PHMB), a broad spectrum disinfectant against many pathogens, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles. The particles formed were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering, electrophoretic mobility, and TEM to measure their morphology and surface chemistry. PHMB-functionalized silver nanoparticles were then evaluated for their antimicrobial activity against a gram-negative bacterial strain, Escherichia coli. These silver nanoparticles were found to have about 100 times higher bacteriostatic and bactericidal activities, compared to the previous reports, due to the combined antibacterial effect of silver nanoparticles and PHMB. In addition to other applications, PHMB-functionalized silver nanoparticles would be extremely useful in textile industry due to the strong interaction of PHMB with cellulose fabrics.

Delignification efficacy of xylanases to facilitate the consequent chemical bleaching of Kraft pulps has been studied widely. In this work, an alkaline and thermally stable cellulase-less xylanase, derived from a xylanolytic Bacillus subtilis, has been purified by a combination of gel filtration and Q-Sepharose chromatography to its homogeneity. Molecular weight of the purified xylanase was 61 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH of 60°C and 8.0, respectively. Xylanase was active in the pH range of 6.0-9.0 and stable up to 70°C. Divalent ions like Ca(2+), Mg(2+) and Zn(2+) enhanced xylanase activity, whereas Hg(2+), Fe(2+), and Cu(2+) were inhibitory to xylanase at 2 mM concentration. It showed K ( m ) and V ( max ) values of 9.5 mg/ml and 53.6 μmol/ml/min, respectively, using birchwood xylan as a substrate. Xylanase exhibited higher values of turn over number (K (cat)) and catalytic efficiency (K (cat)/K (m)) with birchwood xylan than oat spelt xylan. Bleach-boosting enzyme activity at 30 U/g dry pulp displayed the optimum bio-delignification of Kraft pulp resulting in 26.5% reduction in kappa number and 18.5% ISO induction in brightness at 55°C after 3 h treatment. The same treatment improved the pulp properties including tensile strength and burst index, demonstrating its potential application in pre-bleaching of Kraft pulp.

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.

Maternal oxidative stress during pregnancy may impair fetal growth and help in the development of diseases in adulthood. The aim of current study was to assess total oxidation status (TOS), related parameters and their relationship to DNA damage (%) and homocysteine level in normal pregnant women in low-income participants. In a cross-sectional study healthy women were grouped as normal, while age matched nulliparous and singleton pregnancies were included for first, second and third trimester groups. TOS (P<0.01), melanodialdehyde (MDA) (P<0.001), aspartate aminotransferase (AST) (P<0.01), triiodothyronine (T3) (P<0.01), thyroxine (T4) (P<0.01), and homocysteine (P<0.001), in pregnant women were significantly higher as compared to normal healthy women. While serum total proteins (P<0.01), albumin (P<0.01) and total antioxidant status (TAS) (P<0.001) decreased significantly as compared to normal healthy women. Women in third trimester showed a significantly high level of body temperature (P<0.01), triglyceride (P<0.01), LDL-cholesterol (P<0.05), AST (P<0.01), T3 (P<0.01), homocysteine (P<0.001), TOS (P<0.01) and MDA (P<0.001) but a lower concentration of serum proteins, albumin and TAS at the end of the pregnancy. Pearson correlation indicated a positive relationship of homocysteine with triglycerides (P<0.027), TOS (P<0.01), MDA (P<0.035) and had a negative relationship with total protein (P<0.026). DNA damage was strongly related with T3 (P<0.008), TOS (P<0.02), MDA (P<0.037) and MBI (P<0.048) profiles of pregnant women. These changes were considered normal for pregnant women having optimum blood pressure and normal child birth. Hormonal influences and hemodilution may contribute towards the observed changes in this study.

α- and β-Galactosidases find application in food processing, health and nutrition. Aspergillus niger is one of the potent producer of these enzymes and was genotypically improved using gamma-ray induced mutagenesis. The mutant-derivative produced two-fold higher α- and β-galactosidases. For testing genetic variability and its relationship with phenotypic properties of the two organisms, DNA samples of the mutant and parental strains of A. niger were amplified with 28 deca-nucleotide synthetic primers. RAPD analysis showed significantly different pattern between parental and mutant cultures. The mutant derivative yielded homogeneous while parental strain formed heterogeneous amplification patterns. Seven primers identified 42.9% polymorphism in the amplification products, indicating that these primers determined some genetic variability between the two strains. Thus RAPD was found to be an efficient technique to determine genetic variability in the mutant and wild organisms. Both wild and mutant strains were analyzed for their potential to produce galactosidases. Comparison of different carbon sources on enzyme yield revealed that wheat bran is significant (P < 0.01) effective producer and economical source followed by rice bran, rice polishing and lactose. The mutant was significantly better enzyme producer and could be considered for its prospective application in food, nutrition and health and that RAPD can be effectively used to differentiate mutant strain from the parental strain based on the RAPD patterns.

A putative beta-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-beta-D-1-thiogalactopyranoside, the enzyme expressed at approximately 40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A(600) 12 in auto-inducing M9NG medium, beta-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the beta-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45 degrees C and 8.0, respectively, under the assay conditions. K(m) and k(cat) against p-nitrophenyl-beta-D-glucopyranoside were 4 mM and 0.75 sec(-1), respectively. To our knowledge, this is the first report of high-level expression and characterization of a beta-glucosidase from B. halodurans.

Production of xylanases by Thermomyces lanuginosus wild type and its deoxyglucose resistant mutant M7 on different substrates was investigated. The mutation conferred catabolite repression resistance as M7 supported xylanase production on glucose-based medium that was 8.8-fold higher than that of wild type. Product formation parameters were highest in media containing a combination of corncob and corn steep liquor. Among the soluble substrates, xylose was the best inducer. In the presence of glucose, the wild type produced 26 IU of xylanase per g of glucose while the xylanase yield of the mutant was 224 IU per g of glucose. Thermodynamic studies showed that M7 required lower Gibbs free energy, enthalpy and entropy for product formation than the wild type. In biobleaching studies an 18.6% decrease in kappa number and 2.63% increase in brightness for enzyme-treated pulp was observed. Moreover prebleaching with M7-derived enzyme resulted in a 27.3% reduction in chlorine demand as compared with that of 18.5% decrease when wild organism-derived enzyme was employed. These improvements indicate that the mutant-derived enzymes possessed a useful prebleaching potential and could be exploited for large-scale application., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Dependence of plasma total homocysteine (tHcy) and DNA damage profiles on melanodialdehyde (MDA), oxidative stress, liver function tests (LFT), and lipids was studied in non-obese and obese subjects in the Pakistani population. Development of obesity is influenced by both genetic, biochemical and environmental factors. Plasma homocysteine (Hcy) and DNA damage profiles play a pivotal role in its progression. We studied 160 obesity patients and 160 lean subjects. Leukocytes were evaluated for DNA damage by comet assay and blood plasma for biochemical properties using commercial kits. Plasma Hcy level and DNA damage were strongly correlated with triglycerides (P < 0.000), LDL-cholesterol (P < 0.001), systolic blood pressure (P < 0.001), cholesterol (P < 0.004), MDA (P < 0.004) and total oxygen stress (P < 0.004) in obese individuals. Both Hyc and DNA damage were negatively associated with total anti-oxidant response and globulin. Both Hcy profile and DNA damage may have a role in the endothelium damage even in the normal range and are related to triglycerides, ALT, MDA, TOS, HDL- and LDL-cholesterol in the Pakistani population.

The xylanase (Xyn11A) gene (883 bp) was amplified using C. thermophilum DNA as template and cloned into pET-32a (+) and expressed in E. coli BL21 under T(7) promoter. The recombinant xylanase on SDS-PAGE had a molecular mass of 30 kDa. Productivity profiles of xylanase in E. coli recombinant are more than 4-fold of that produced from T. reesei RUTC-30, 5-fold of that produced by the donor and significantly higher than the values reported on other E. coli, and Saccharomyces cerevisiae recombinants. Temperature stability, pH stability, and other kinetic parameters confirmed that the gene product was thermo-stable in alkaline buffer favoring its suitability to bio-bleaching of kraft pulp.

Aims: To investigate the effect of molasses concentration, initial pH of molasses medium, and inoculum's size to maximize ethanol and minimize methanol, fusel alcohols, acetic acid and aldehydes in the fermentation mash in industrial fermentors., Methods and Results: Initial studies to optimize temperature, nitrogen source, phosphorous source, sulfur supplement and minerals were performed. The essential nutrients were urea (2 kg in 60 m(3)), 0.5 l each of commercial phosphoric acid and sulfuric acid (for pH control) added at the inoculum preparation stage only. Yields of ethanol, methanol, fusel alcohols, total acids and aldehydes per 100-l fermentation broth were monitored. Molasses at 29 degrees Brix (degree of dissolved sugars in water), initial pH 4.5, inoculum size 30% (v/v) and anaerobic fermentation supported maximum ethanol (7.8%) with Y(P/S) = 238 l ethanol per tonne molasses (96.5% yield) (8.2% increase in yield), and had significantly lower values of byproducts than those in control experiments., Conclusions: Optimization of process variables resulted in higher ethanol yield (8.2%) and reduced yield of methanol, fusel alcohols, acids and aldehydes., Significance and Impact of the Study: More than 5% substrate is converted into byproducts. Eliminating or reducing their formation can increase ethanol yield by Saccharomyces cerevisiae, decrease the overall cost of fermentation process and improve the quality of ethanol.

A 2-deoxyglucose-resistant mutant (M7) of Humicola lanuginosa was obtained by exposing conidia to γ-rays and permitting expression in broth containing 0.6% 2-deoxyglucose (DG) and cellobiose (1%) before plating on DG esculin-ferric ammonium citrate agar medium from which colonies showing faster and bigger blackening zones were selected. Kinetic parameters for enhanced ß-glucosidase (BGL) synthesis by M7 were achieved when corncobs acted as the carbon source. The combination between corncobs and corn steep liquor was the best to support higher values of all product formation kinetic parameters. Effect of temperature on the kinetic and thermodynamic attributes of BGL production equilibrium in the wild organism and M7 was studied using batch process at eight different temperatures in shake-flask studies. The best performance was found at 45°C and 20 g L(-1) corncobs in 64 h. Both growth and product formation (17.93 U mL(-1)) were remarkably high at 45°C and both were coupled under optimum working conditions. Product yield of BGL from the mutant M7 (1556.5 U g(-1) dry corncobs) was significantly higher than the values reported on all fungal and bacterial systems. Mutation had thermo-stabilization influence on the organism and mutant required lower activation energy for growth and lower magnitudes of enthalpy and entropy for product formation than those demanded by the wild organism, other mesophilic and thermo-tolerant organisms. In the inactivation phase, the organisms needed lower values of activation energy, enthalpy and entropy for product formation equilibrium, confirming thermophilic nature of metabolic network possessed by the mutant organism.

Bacillus strain CTP-09 yielded maximum productivity (1120 IU/L.h) of extracellular endoglucanase (CMCase) on 0.5% cellobiose after 10 h fermentation at 55 degrees C. The purified enzyme is mono-meric in nature and exhibits stability up to 80 degrees C and over a pH range (6.0-9.0). Activation energy, enthalpy and entropy of catalysis, and inactivation indicated that this CMCase is highly thermos-table. Purified enzyme possessed high power of defibrillation of textile and was minutely inhibited by anionic detergent and oxidizing agent comparable with inhibition by commercial enzyme. This polypeptide could be exploited for mass production and application in local industries.

Kinetic parameters for production of beta-xylosidase by Kluyveromyces marxianus were determined in growth media containing glucose, xylose, cellobiose, sucrose and lactose as carbon sources. K. marxianus achieved maximum beta-xylosidase specific product yield (Y(P/X)) when grown on xylose. Basal level of activity was achieved in cultures grown on glucose. Kinetic parameters of enzyme production and cell mass formation were correlated. Enzyme synthesis was regulated by an induction mechanism and growth-dependent repression mechanism. Thermodynamic analysis revealed that the cell system exerted protection against thermal inactivation. A partially purified enzyme showed good stability when incubated at 60 degrees C and was quite stable at a pH of 5.0-7.0 and may be exploited for commercial applications.

Purified endoglucanase from C. biazotea mutant 51SM(r) was successfully immobilized on Eudragit L-100, with 75 % yield of immobilization. This method improved the kinetic and thermodynamic properties of the enzyme. Immobilization significantly decreased entropy and enthalpy of inactivation of biocatalyst and made it functionally and thermodynamically more stable and reusable compared to free one.

Single cell protein was produced from the defatted rice polishings by fermentation with Candida utilis in an aerated 14-L fermentor to optimize bioprocess variables. Maximum values of specific growth rate coefficient (mu, h(-1)), cell mass yield (Y(X/S), g/g) and cell mass productivity (g/Lh) were 0.31, 0.65, and 1.24, respectively under optimized conditions of aeration rate (1 v.v(-1) m(-1)), dissolved oxygen (50%), corn steep liquor (5%), temperature (35 degrees C), and substrate concentration (90 g rice polishings/L) in yeast salt medium (pH 6.0). The kinetic parameters for 50-L fermentor under same conditions were 0.33 h(-1), 0.66 g/g, 1.33 g/Lh, 2.25 g/Lh, 1.23 g/Lh, 0.45 g/g substrate and 0.20 g/g cell h for mu, Y(X/S), Q(X), Q(S), Q(CP), Y(TP/S), and q(CP), respectively and were significantly higher than their respective values reported on C. utilis in batch culture studies. This biomass protein contained 23.6%, 32.75%, 11.50%, 12.95%, 10.5%, and 0.275% true protein, crude protein, crude fiber, ash, cellulose and RNA content respectively. This implied that the fermentation process could be up scaled to manufacture animal feed. Gross metabolizable energy content of dried SCP was 29,711 kcal/kg and indicated that the SCP could serve both as energy as well as a protein source. Yeast can replace expensive feed ingredients currently being incorporated in poultry feed and can reduce cost of poultry ration by 0.33 US dollars-0.51 US dollars/100 kg bag and improve the economics of feed production in our country.

Purified glucoamylase from Arachniotus citrinus was immobilized on polyacrylamide gel with 70% yield of immobilization. The immobilization improved the pH optima, temperature optima, values of K(m), V(max), and activation energy. Irreversible thermal denaturation studies of soluble and immobilized glucoamylase indicated that immobilization decreased the entropy and enthalpy of deactivation by magnitudes and made the immobilized glucoamylase thermodynamically more stable.

Uricase from bovine kidney, purified to homogeneity level, had a molecular weight of 70 kDa. The apparent K(m) and V(max) values for uric acid hydrolysis were 0.125 mM and 102 IU mg(-1) protein respectively. The activation energy requirement for uric acid hydrolysis by uricase and inactivation of enzyme were 11.6 and 14.5 kJ/M respectively. Both enthalpy (Delta H*) and entropy of activation (Delta S*) for uricase activity were lower than those reported for some thermostable enzymes.

Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-dopa from L-tyrosine using chemical mutation. Different putative mutant strains of the organism were tested for the production of L-dopa in triplicate shake-flask cultures. Among these putative mutants, the strain designated SI-12 gave a maximal production of L-dopa (444+/-14 mg of L-dopa/g of cells). The regulation of L-dopa from different carbon source solutions [initial substrate concentration (S(0))=30 g/l] by the mutant culture was investigated. At an initial pH (pH(0)) of 5.0 and a temperature (T) of 30 degrees C, 100% of sugars were utilized for product and cell mass formation, corresponding to final L-dopa product yield of 189+/-8 mg/g of substrate utilized and maximum volumetric and specific productivities of 145+/-5 mg/h per litre and 155+/-8 mg/h per g of cells respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is, to our knowledge, the highest reported in the literature. To explain the kinetic mechanism of L-dopa formation and its thermal inactivation, the thermodynamic parameters were determined with the application of the Arrhenius model. Activation enthalpy and entropy for product formation, in the case of the mutant derivative, were 40 kJ/mol and 0.076 kJ.mol(-1).K(-1) for its production and 116 kJ/mol and 0.590 kJ.mol(-1).K(-1) for thermal inactivation respectively. The respective values for product formation and product de-activation were lower than the respective values for the parental culture. Therefore the mutant strain was thermodynamically more resistant to thermal denaturation during the product-formation process.

The mutation, conferring streptomycin and deoxyglucose resistance on cells, had profound effect on the kinetic and thermodynamic parameters inferring thermostabilization of beta-glucosidase from mutant 51 SM(r) of Cellulomonas biazotea. Free energy of activation for substrate binding, enthalpy and entropy of activation for irreversible denaturation of mutant-derived enzyme were decreased compared with enzyme from wild organism suggesting that the mutation partly stabilized the enzyme and that mutation made it more reactive.

Two isoenzymes of endo-1,4-beta-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a combination of ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel filtration chromatography. The native mol wts of the least acidic xylanase (LAX) and the highly acidic xylanase (HAX) were 25 and 144 kDa and the subunit mol wts were 25 and 36 kDa, respectively. The kcat values of LAX and HAX for oat-spelt xylan at 40 degrees C, pH 6.5, were 95,000 and 9900 min-1 and the Km values of LAX and HAX were 30 and 3.3 mg/mL. The thermodynamic activation parameters of xylan hydrolysis showed that the high activity of LAX when compared with HAX was not owing to a reduction in DeltaH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50 degrees C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (DeltaH#=306 kJ/mol) compared with HAX (DeltaH#=264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (DeltaS*=-232 J/[mol.K]) was more thermostable than HAX (DeltaS*=490 J/[mol.K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.

Aims: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor., Methods and Results: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1.45-fold improved than its wild parent with respect to ethanol productivity (7.2 g l-1 h-1), product yield (0.44 g ethanol g-1 substrate utilized) and specific ethanol yield (19.0 g ethanol g-1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P

The influence of carbon and nitrogen sources on the production of cellulases was investigated. The enzyme production was variable according to the carbon source. Levels of beta-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates. The enzyme is subject to carbon source control by easily metabolizable sugars. Wheat bran and cellulose were the most effective promoters of beta-cellobiohydrolase and filter paperase (FPase) activities respectively, followed by rice bran. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of A. niger growing on wheat bran. In defined medium with cellobiose, the cellobiohydrolase titres were 2- to 110-fold higher with cells growing on monomeric sugars and 1.5 times higher than cells growing on other disaccharides. It appeared that synthesis of beta-cellobiohydrolase varied under an induction mechanism, and a repression mechanism which changed the rate of synthesis of beta-cellobiohydrolase and FPase in induced over non-induced cultures. In this organism, substantial synthesis of beta-cellobiohydrolase can be induced by cellobiose, cellodextrin, cellulose or cellulose and hemi-cellulose containing substrates which showed low volumetric substrate uptake rate. The organism required limiting concentration of carbon, nitrogen or phosphorous for production of beta-cellobiohydrolase and FPase. During growth of A. niger on wheat bran, maximum volumetric productivities (Qp) of beta-cellobiohydrolase and FPase were 39.6 and 32.5 IU/lh and were significantly higher than the values reported for some other potent fungi and bacteria. The addition of actinomycin D (a repressor of transcription) and cycloheximide, (a repressor of translation) completely repressed CBH/FPase biosynthesis, suggested that the regulation of CBH synthesis in this organism occurs at both transcriptional and translational level. Thermodynamic studies revealed that the culture exerted protection against thermal inactivation when exposed to different fermentation temperatures.

The maximum product yield of endo-glucanase (650 IU g(-1) substrate) from Cellulomonas biazotea mutant 51 Sm(r) was 1.5- to 2.5-fold more than was produced by the wild type cells and was twice that reported by previous researchers. Mutation substantially improved the enthalpy (DeltaH (*)) and entropy of activation (DeltaS (*)) for product formation, turnover number, specificity constant activation energy, free energies for transition state formation and substrate binding for CMC hydrolysis respectively.

The maximum volumetric productivity of beta-galactosidase by a Kluyveromyces marxianus mutant, grown on lactose/corn steep liquor medium for 3 d, was 150 IU l(-1) h(-1) which is twice that of the parent organism. During product formation, mutated cells provided more resistance against thermal inactivation.

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on beta-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and beta-glucosidase production rate. The highest product yield, specific product yield, and productivity of beta-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved beta-glucosidase production measured as product yield (YP/S) and volumetric productivity (QP) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L.h]) of beta-glucosidase occurred on cellobiose in the presence of CSL at 35 degrees C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of beta-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60 degrees C and may find application in some industrial processes.

The highest productivity (20 IU l(-1) h(-1)) of beta-glucosidase by a mutant of Cellulomonas biazotea was 2.5-fold more than that of the parent organism. The enzyme had a lower activation energy (57 kJ mol(-1)) than the native enzyme (68 kJ mol(-1)). The enzyme from the mutant had enthalpy and entropy values for irreversible intactivation of 95.6 kJ mol(-1) and 60 J.mol(-1) K(-1) compared with 108 kJ mol(-1) and 86 J mol(-1) K(-1) for the native enzyme suggesting that the mutation had stabilized the enzyme.

Aims: The present investigation deals with the effect of raw starch hydrolyzing glucoamylase by a derepressed mutant of Aspergillus niger on enhanced productivity of ethanol from uncooked starch under non-aseptic conditions., Methods and Results: The parental culture of Aspergillus niger was improved using gamma-ray treatment. One derepressed mutant was isolated after extensive screening and optimization and grown on corn cobs, maize starch, soluble starch and wheat bran solid media moistened with Vogel's salts solution and corn steep liquor. The mutant was 2.5-fold improved over its parent with respect to enzyme productivity, product yield and specific activity. The enzyme from mutated culture was also improved for enzyme properties and could effectively hydrolyze raw starch without the aid of alpha-amylase. Starch hydrolyzed with mutant-derived glucoamylase supported higher volumetric and product yields of ethanol than those of parental and other strains., Significance and Impact of the Study: The results of the present study are of commercial value. Ethanol product yield coefficient, and volumetric productivity revealed the hyper-productivity of ethanol from raw starch hydrolyzate obtained with mutant-derived glucoamylase without addition of liquefying alpha-amylase under non-aseptic conditions.

The mutation had dramatic effect on the kinetic and thermodynamic parameters inferring thermostability of endo-glucanase from Cellulomonas biazotea mutant 51 SM(r). The denaturation activation energies of native and mutated enzymes were 73.3 and 68.8 kJ/mol respectively. They showed compensation effect at 55 degrees C. Both enthalpy and entropy values of irreversible thermal inactivation for mutated enzyme were decreased suggesting that the mutation partly stabilized the enzyme.

Purified extracelluar glucoamylase from Arachniotus sp. was used for kinetic and thermodynamic characterization. Thermal inactivation followed first order kinetics. The denaturation/activation energies of enzyme were 57 and 89 kJ mol(-1), respectively. Both enthalpy and entropy of activation for inactivation were lower than those for glucoamylases reported in literature. It is suggested that the enzyme is highly thermostable and is suitable for industrial applications.

The maximum productivity of beta-glucosidase by Saccharomyces cerevisiae recombinants under the control of GALI promoter was 100 IU l(-1) h(-1). The highest productivity of beta-glucosidase by a S. cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g(-1) cellobiose and g ethanol l(-1) h(-1), respectively.

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.

Saccharomyces cerevisiae and Candida tropicalis were used separately and as co-culture for simultaneous saccharification and fermentation (SSF) of 5-20% (w/v) dry corn cobs. A maximal ethanol concentration of 27, 23, 21 g/l (w/v) from 200 g/l (w/v) dry corn cobs was obtained by S. cerevisiae, C. tropicalis and the co-culture, respectively, after 96 h of fermentation. However, theoretical yields of 82%, 71% and 63% were observed from 50 g/l dry corn cobs for the above cultures, respectively. Maximal xylitol concentration of 21, 20 and 15 g/l from 200 g/l (w/v) dry corn cobs was obtained by C. tropicalis, co-culture, and S. cerevisiae, respectively. Maximum theoretical yields of 79.0%, 77.0% and 58% were observed from 50 g/l of corn cobs, respectively. The volumetric productivities for ethanol and xylitol increased with the increase in substrate concentration, whereas, yield decreased. Glycerol and acetic acid were formed as minor by-products. S. cerevisiae and C. tropicalis resulted in better product yields (0.42 and 0.36 g/g) for ethanol and (0.52 and 0.71 g/g) for xylitol, respectively, whereas, the co-culture showed moderate level of ethanol (0.32 g/g) and almost maximal levels of xylitol (0.69 g/g).

Carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 was purified by a combination of ammonium sulphate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography on FPLC with 9-folds increase in specific activity. Native and subunit molecular weights were found to be 36 kDa each. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). Similarly, the enzyme was modified by EDC in the presence of ethylenediamine dihydrochloride plus cellobiose for 75 min (EDAM75). The neutralization (GAM15 and GAM75) and reversal (EDAM75) of negative charges of carboxyl groups of CMCase had profound effect on the specificity constant (k(cat)/K(m)), pH optima, pK(a)'s of the active-site residues and thermodynamic parameters of activation. The specificity constants of native, GAM15, GAM75, and EDAM75 were 143, 340, 804, and 48, respectively. The enthalpy of activation (DeltaH(#)) of Carboxymethylcellulose (CMC) hydrolysis of native (50 and 15 kJ mol(-1)) and GAM15 (41 and 16 kJ mol(-1)) were biphasic whereas those of GAM75 (43 kJ mol(-1)) and EDAM75 (41 k J mol(-1)) were monophasic. Similarly, the entropy of activation (DeltaS(#)) of CMC hydrolysis of native (-61 and -173 J mol(-1) K(-1)) and GAM15 (-91 and -171 J mol(-1) K(-1)) were biphasic whereas those of GAM75 (-82 J mol(-1) K(-1)) and EDAM75 (-106 J mol(-1) K(-1)) were monophasic. The pH optima/pK(a)'s of both acidic and basic limbs of charge neutralized CMCases increased compared with those of native enzyme. The CMCase modification in the presence of glycinamide and absence of cellobiose at different pH's periodically activated and inhibited the enzyme activity indicating conformational changes. We believe that the alteration of the surface charges resulted in gross movement of loops that surround the catalytic pocket, thereby inducing changes in the vicinity of active site residues with concomitant alteration in kinetic and thermodynamic properties of the modified CMCases.

Genes for beta-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotea, were cloned in pUC18 in its SacI cloning site and transformed to E. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representative E. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of beta-glucosidase on E. coli. Expression of the bgl genes resulted in overproduction of beta-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carrying bgl genes from the representative recombinant E. coli were isolated with SacI, ligated in the shuttle vector pYES 2.0 in its SacI site and transformed to E. coli and S. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of beta-glucosidase on S. cerevisiae and enabled it to grow on cellobiose and salicin. The gall promoter of shuttle vector pYES 2.0 enabled the organisms to produce twice more beta-glucosidase than that supported by the lacZ-promoter of pUC18 plasmid in E. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains of S. cerevisiae. The enzyme produced by bgl+ yeast and E. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the beta-glucosidases from S. cerevisiae were substantially the same as those from C. biazotea.

Purified beta-glucosidase fromCellulomonas biazotea had an apparentK (m) andV for 2-nitrophenyl beta-D: -glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of beta-glucosidase was 65 kJ/mol. The inhibition by Mn(2+) vs. oNPG of parental beta-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 micromol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK (a)'s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of beta-glucosidase decreased in the presence of 10 % ethanol. The half-life of beta-glucosidase in 1.75 mol/L urea at 35 degrees C was 145 min, as determined by 0-9 mol/L transverse urea gradient-PAGE.

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing alpha-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and beta-glucoside hydrolyzing activities. All strains synthesized beta-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on alpha-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10-13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. beta-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and beta-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi.