Synthesis of L-asparaginase bySerratia marcescens (Nima)

The production of L-asparaginase by two mutants ofSerratia marcescens grown on 14 different media was studied. The enzyme content increased from trace levels to 2.4 international units per ml when the organisms were grown in glycerol-peptone yeast extract medium. Glucose was the best carbon source under aerobic conditions. The enzyme content increased when L-asparagine was present in the growth medium.     

Enhanced Biosynthesis of Sulfide Oxidase by Arthrobacter Species Using Response Surface Methodology

Response surface methodology was used to develop a fermentation medium for the enhanced biosynthesis of a novel sulfide oxidase by Arthrobacter species strain FR‐3. The interactive effect of the medium components – such as glucose as the carbon source, and tryptone and yeast extract as the nitrogen source – was evaluated by a 2³‐factorial central composite statistical design. Glucose and yeast extract were found to be the more influential medium constituents compared to tryptone since they had lower coefficients of linear effect, P‐values (< 0.02). The optimal fermentation medium components for the enhanced production of sulfide oxidase were recorded as glucose (8.98 g/L), tryptone (10.62 g/L) and yeast extract (7.3 g/L). Optimization of the medium constituents increased the experimental enzyme yield by 54 % compared to the unoptimized medium. This is the first report on the overproduction of sulfide oxidase by using response surface methodology.     

High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium

To develop an economical industrial medium, untreated cane molasses (UCM) was tested as a carbon source for fermentation culturing of Escherichia coli. To test the industrial application of this medium, we chose a strain co-expressing a carbonyl reductase (PsCR) and a glucose dehydrogenase (BmGDH). Although corn steep liquor (CSL) could be used as an inexpensive nitrogen source to replace peptone, yeast extract could not be replaced in E. coli media. In a volume of 40 ml per 1-l flask, a cell concentration of optical density (OD₆₀₀) 15.1 and enzyme activities of 6.51 U/ml PsCR and 3.32 U/ml BmGDH were obtained in an optimized medium containing 25.66 g/l yeast extract, 3.88 g/l UCM, and 7.1% (v/v) CSL. When 3.88 g/l UCM was added to the medium at 6 h in a fed-batch process, the E. coli concentration increased to OD₆₀₀ of 24, and expression of both PsCR and BmGDH were twofold higher than that of a batch process. Recombinant cells from batch or fed-batch cultures were assayed for recombinant enzyme activity by testing the reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE). Compared to cells from batch cultures, fed-batch cultured cells showed higher recombinant enzyme expression, producing 560 mM CHBE in the organic phase with a molar yield of 92% and an optical purity of the (S)-isomer of >99% enantiomeric excess.     

Glucoamylase production by solid-state fermentation using rice flake manufacturing waste products as substrate

Glucoamylase production has been investigated by solid-state fermentation of agro-industrial wastes generated during the processing of paddy to rice flakes (categorized as coarse, medium and fine waste), along with wheat bran and rice powder by a local soil isolate Aspergillus sp. HA-2. Highest enzyme production was obtained with wheat bran (264 +/- 0.64 U/gds) followed by coarse waste (211.5 +/- 1.44 U/gds) and medium waste (192.1 +/- 1.15 U/gds) using 10(6) spores/ml as inoculum at 28 +/- 2 degrees C, pH 5. A combination of wheat bran and coarse waste (1:1) gave enzyme yield as compared to wheat bran alone. Media supplementation with carbon source (0.04 g/gds) as sucrose in wheat bran and glucose in coarse and medium waste increased enzyme production to 271.2 +/- 0.92, 220.2 +/- 0.75 and 208.2 +/- 1.99 U/gds respectively. Organic nitrogen supplementation (yeast extract and peptone, 0.02 g/gds) showed a higher enzyme production compared to inorganic source. Optimum enzyme activity was observed at 55 degrees C, pH 5. Enzyme activity was enhanced in the presence of calcium whereas presence of EDTA gave reverse effect.     

Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen-printed electrode

A disposable and mediatorless immunosensor based on a conducting polymer (5,2':5'2"-terthiophene-3'-carboxylic acid) coated screen-printed carbon electrode has been developed using a separation-free homogeneous technique for the detection of rabbit IgG as a model analyte. Horseradish peroxidase (HRP) and streptavidin were covalently bonded with the polymer on the electrode and biotinylated antibody was immobilized on the electrode surface using avidin-biotin coupling. This sensor was based on the competitive assay between free and labeled antigen for the available binding sites of antibody. Glucose oxidase was used as a label and in the presence of glucose, H(2)O(2) formed by the analyte-enzyme conjugate was reduced by the enzyme channeling via HRP bonded on the electrode. The catalytic current was monitored amperometrically at -0.35 V vs. Ag/AgCl and this method showed a linear range of RIgG concentrations from 0.5 to 2 microg/ml with standard deviation +/-0.0145 (n=4). Detection limit was determined to be 0.33 microg/ml.     

A media design program for lactic acid production coupled with extraction by electrodialysis

The aim of this study was to investigate industrial media for lactic acid fermentation to reduce the cost of nitrogen sources. Corn steep liquor (CSL) was successfully used at 5% (v/v) in batch fermentations. Use of soluble CSL improved the productivity approximately 20% with an advantage of clearer fermentation broth. Yeast extract (YE)-complemented CSL media further increased the productivity. It was found that 3.1 g L(-1) yeast extract and 5% CSL could be an effective substitute for 15 g L(-1) yeast extract in 10% glucose medium. Spent brewery yeast was also used as a sole nitrogen source equivalent to 5% CSL. Lactic acid was recovered by electrodialysis from the cell free broth. Depleted cell free broth supplemented with 5 g L(-1) of yeast extract performed reasonably in batch cultures. Reuse of the fermentation broth may reduce the cost of raw materials as well as minimize the fermentation wastes.     

Production of dihydrofolate reductase by cloned Escherichia coli and its application to asymmetric synthesis of l-leucovorin.

We have investigated culture conditions for production of dihydrofolate reductase by Escherichia coli harboring a high expression plasmid, pTP64-1. Sorbitol addition and pH control were effective for the production of the enzyme in a jar fermentor. The enzyme was purified from a cell-free extract by column chromatographies on DEAE-Cellulofine and Superose Prep12 and showed a single band on SDS-polyacrylamide gel electrophoresis. The reduction of 200 mM dihydrofolate to 6(S)-tetrahydrofolate, an intermediate for l-leucovorin synthesis, was complete in 2 hr under anaerobic conditions, using 1.5 units/ml of the purified enzyme.     

Effects of reactive oxygen metabolites on erythropoietin production in renal carcinoma cells

The present studies were undertaken to determine the effects of reactive oxygen metabolites on erythropoietin (Ep) biosynthesis in Ep-producing renal carcinoma (RC) cells using a sensitive radioimmunoassay for Ep. Xanthine (10-5M) and increasing concentrations of xanthine oxidase (8 x 10(-7) to 5 x 10(-4) units/ml) produced a significant dose-related increase in Ep production at a concentration of greater than or equal to 4 x 10(-6) units/ml, whereas xanthine alone had no effect. Catalase, a scavenger of hydrogen peroxide (H2O2), in concentrations of 50 to 500 micrograms/ml produced a significant inhibition of the increase in Ep production induced by xanthine-xanthine oxidase; while no effect was seen on basal levels of Ep production and the growth of RC cells. Glucose oxidase (greater than or equal to 0.032 mU/ml), a direct H2O2 generator, and exogenous H2O2 (greater than or equal to 4 x 10(-6)M) added to the incubation mixture, caused a significant enhancement of Ep production in a dose-dependent manner. Xanthine-xanthine oxidase, glucose oxidase, and H2O2 in the above concentrations did not produce significant cytotoxicity (51Cr release or trypan blue dye exclusion). The present data suggests that H2O2, a reactive oxygen metabolite may play a significant role in Ep production.     

High-density spore production of a <Emphasis Type="Italic">B</Emphasis>. <Emphasis Type="Italic">cereus</Emphasis> aquaculture biological agent by nutrient supplementation

Previous studies have demonstrated the efficacy of our Bacillus cereus isolate (NRRL 100132) in reducing concentrations of nitrogenous wastes and inhibiting growth of fish pathogens. In vivo efficacy and tolerance to a range of physiological conditions in systems used to rear Cyprinus carpio make this isolate an excellent candidate for aquaculture applications. Production cost is an important consideration in development of commercially relevant biological products, and this study examines the optimization of nutrient supplementation, which has an impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was identified as a lower cost and more effective nutrient source in comparison to conventional nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was modeled to develop a tool for optimization of nutrient concentration in fermentation. An excellent fit of the model was confirmed in laboratory fermentation studies. A cost comparison revealed that production using liquid phytase and ultrafiltered-treated CSL was less expensive than spray-dried CSL and supported cultivation of B. cereus spores at densities higher than 1 × 10¹⁰ CFU ml⁻¹.     

INFLUENCE OF GLUCOSE OXIDASE ON THE GROWTH OF ESCHERICHIA COLI 0157:H7, LISTERIA MONOCYTOGENES, AND SALMONELLA TYPHIMURIUM IN UNIVERSAL PREENRICHMENT BROTH

Glucose oxidase (GO), a food-grade enzyme, was compared with Oxyrase^TM oxygen reducing membrane fraction in Universal Preenrichment Broth (UPB) for enhancement of the growth of the facultatively anaerobic pathogens Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes Scott A. Oxidation-reduction potential (ORP) and pH changes in UPB following the addition of GO (4 units/ml) or Oxyrase^TM (0.5 units/ml) were measured. Microbial growth was evaluated at 0, 3, 6, 18, and 24 h of incubation using spiral plating. Nonenzyme supplemented UPB served as the control. Oxyrase^TM provided a higher oxygen scavenging action in terms of ORP decrease during the initial 6 h of incubation. However, no difference occurred in Eh between Oxyrase^TM and GO by 18 h, with both enzyme systems effectively reducing the Eh compared to that of the control. A 1.0 pH unit reduction was observed in GO-supplemented UPB after 18 h, indicating production of gluconic acid. The pH decrease in Oxyrase^TM - supplemented media was 0.2 units. By 6 h, the E. coli O157:H7 population was enhanced by 0.6 and 1.4 log CFU/ml in Oxyrase^TM -supplemented media, compared to the control and GO-supplemented media, respectively. By 18 h, 0.4 and 0.9 log CFU/ml growth enhancements of the E. coli O157:H7 populations were seen in GO- and Oxyrase^TM -supplemented media, respectively, compared to the control. By the end of 18 h, counts of S. typhimurium and L. monocytogenes increased by 0.6 and 0.2 log units, respectively, in GO-supplemented media compared to the control.     

Characterization of an exceedingly active NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima

An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80 degrees C. The V(max) was 230 +/- 14 mumol/min/mg (k(cat)/K(m) = 548,000 min(-1) mM(-1)), and the K(m) values for NADH and oxygen were 42 +/- 3 and 43 +/- 4 muM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima.     

Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill-defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch-to-batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL-based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL-glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.     

Synthesis of Protocatechuate Oxygenase by Pseudomonas fluorescens in the Presence of Exogenous Carbon Sources.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Synthesis of protocatechuate oxygenase by Pseudomonas fluorescens in the presence of exogenous carbon sources. J. Bacteriol. 90:15-22. 1965.-The addition of glucose, ribose, or fructose (0.45 or 45.0 mumoles/ml) simultaneously with protocatechuic acid shortens the lag period required for synthesis of protocatechuate oxygenase by a washed-cell suspension of Pseudomonas fluorescens. Glucose is readily oxidized and supports growth of P. fluorescens, whereas neither ribose nor fructose readily supports growth. High glucose concentrations (45.0 mumoles/ml) shorten the lag period but lower the total enzyme synthesis. The pH drops during glucose oxidation, and this is accompanied by a decrease in the rate of enzyme synthesis. High glucose concentrations, with adequate buffering, permitted "normal" enzyme synthesis. A decrease in the total enzyme synthesis was not observed in the presence of high concentrations of ribose or fructose. Succinate, pyruvate, acetate, or formate (0.45 mumole/ml) were readily oxidized, but did not shorten the lag period required for synthesis of the enzyme. The data suggest that glucose, ribose, or fructose may serve as a "specific" carbon source (such as ribose-5-phosphate or a similar precursor important in ribonucleic acid synthesis) functional in the synthesis of protocatechuate oxygenase.     

Postnatal Development and Isolation of Peroxisomes from Brain

We analyzed the postnatal peroxisome development in rat brain by measuring the enzyme activities of catalase and acyl-CoA oxidase and beta-oxidation of [1-14C]lignoceric acid. These enzyme activities were higher between 10 and 16 days of postnatal life and then decreased. We developed and compared two different methods for isolation of enriched peroxisomes from 10-day-old rat brain by using a combination of differential and density gradient centrifugation techniques. Peroxisomes in Percoll (self-generating gradient) banded at a density of 1.036 +/- 0.012 g/ml and in Nycodenz continuous gradient at 1.125 +/- 0.014 g/ml. Acyl-CoA oxidase, D-amino acid oxidase, L-pipecolic acid oxidase, and dihydroxyacetone phosphate acyltransferase activities and activities for the oxidation of very long chain fatty acid (lignoceric acid) were almost exclusively associated with catalase activity (a marker enzyme for peroxisomes) in the gradient. The postnatal increase in peroxisomal activity with the onset of myelination and the presence of enzyme for the biosynthesis of plasmalogens and oxidation of very long chain fatty acid (both predominant constituents of myelin) suggest that brain peroxisomes may play an important role in the assembly and turnover of myelin.     

Hyperproduction of N-acetylneuraminate lyase by the gene-cloned strain ofEscherichia coli

Summary Optimal culture medium for production ofN-acetylneuraminate lyase (NANA lyase) by the gene-cloned strain ofEscherichia coli, E. coli(pNALl), was screened to develop the process for the industrial production of NANA lyase. Out of nutrients tested, corn steep liquor (CSL) and sugarcane molasses were superior nutrient sources for the enzyme production. Hyperproduction (6–8 units/ml-broth) was achieved on the medium consisted of CSL and molasses.     

Fermentative production of succinic acid from glucose and corn steep liquor byAnaerobiospirillum succiniciproducens

Anaerobiospirillum succiniciproducens requires expensive complex nitrogen sources such as yeast extract and polypeptone for its growth and succinic acid production. It was found thatA. succiniciproducens was able to grow in a minimal medium containing glucose when supplemented with corn steep liquor (CSL) as the sole complex nitrogen source. The concentration of CSL had a significant effect on the glucose consumption byA. succiniciproducens. When 10–15 g/L of CSL was supplemented, cells were grown to an OD₆₆₀ of 3.5 and produced 17.8 g/L succinic acid with 20 g/L glucose. These results are similar to those obtained by supplementing yeast extract and polypeptone, thereby suggesting that succinic acid can be produced more economically using glucose and CSL.     

Characterization and evaluation of corn steep liquid in acetone-butanol-ethanol production by Clostridium acetobutylicum

Corn steep liquor (CSL) obtained from a commercial starch manufacturing process was analyzed and tested as a complex nutrient source for ABE (acetone, butanol, and ethanol) production by Clostridium acetobutylicum PJC4BK_AdhE1. CSL contained a wealth of trace elements and nitrogenous components, proteins and amino acids. ABE production increased the content of CSL was raised up to 6% (v/v) in medium and then decreased at higher contents. In 6% CSL-containing medium, C. acetobutylicum PJC4BK_AdhE1 produced 21.4 g/L of ABE with a yield of 0.41 g/g in 18 h of fermentation. Although the final titer of ABE was similar in CSL containing media and Clostridial Growth Medium (CGM, containing yeast extract and asparagines as complex nutrients), the yield and productivity of ABE in 6% CSL-containing medium were found to be higher than 32 and 26%, respectively.     

Sorghum straw for xylanase hyper-production by Thermomyces lanuginosus (D2W3) under solid-state fermentation

This paper reports the production of very high levels of cellulase free xylanase and associated hemicellulases by an indigenous thermophilic isolate of Thermomyces lanuginosus (D(2)W(3)) using solid-state fermentation. Sorghum straw, an inexpensive and abundant source of carbon supported maximal xylanase activity (11,855 units/g dry substrate). Culturing T. lanuginosus D(2)W(3) on sorghum straw and optimizing other culture conditions (media types, particle size of carbon source, inoculum level, inoculum age and additives), yielded increased levels of xylanase (39,726 units/g dry substrate). Further optimization of enzyme production was carried out using Box-Behnken design of experiments with three independent variables (inoculum level, glycerol and ammonium sulphate concentrations) which resulted in very high levels of xylanase, 48,000+/-1774 units/g dry substrate, and 2.6+/-0.2, 13.4+/-0.56, 68+/-1.7, 1.4+/-0.08, 1.2+/-0.05 (units/g dry substrate) of beta-xylosidase, alpha-galactosidase, pectinase, beta-mannosidase and alpha-L-arabinofuranosidase, respectively.     

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.     

Increased glucose metabolism by enzyme-loaded erythrocytes in vitro and in vivo normalization of hyperglycemia in diabetic mice.

The main metabolic properties of human red blood cells (RBC) overloaded with glucose catabolizing enzymes such as hexokinase and glucose oxidase were evaluated. Human erythrocytes loaded with human hexokinase metabolized 3.1 +/- 0.2 mumol/h/ml RBC of glucose, an amount double that consumed by normal and unloaded cells (1.46 +/- 0.16 mumol/h/ml RBC), while glucose oxidase-loaded erythrocytes consumed up to 5.5 +/- 0.5 mumol/h/ml RBC of glucose but with a time-dependent increase in methemoglobin formation due to the H2O2 produced in the glucose oxidase reaction. This methemoglobin production was greatly reduced while glucose consumption was increased (8.1 +/- 0.4 mumol/h/ml RBC) by coentrapment of hexokinase and glucose oxidase. Similar results were obtained in mouse red blood cells, although the role of hexokinase was less pronounced due to a higher basal level of this enzyme. When administered to diabetic mice the hexokinase/glucose oxidase-overloaded erythrocytes had a circulating half-life of 5 days and were able to regulate blood glucose at near physiological levels. A single intraperitoneal administration of 500 microliters of enzyme-loaded cells maintained a near-normal blood glucose concentration for 7 +/- 1 days, while repeated administrations at 10-day intervals were effective in the regulation of blood glucose levels for several weeks. These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way to reduce abnormally elevated blood glucose.