Production and application of amylases of Rhizopus oryzae and Rhizopus microsporus var. oligosporus from industrial waste in acquisition of glucose

Amylases from Rhizopus oryzae and Rhizopus microsporus var. oligosporus were obtained using agro-industrial wastes as substrates in submerged batch cultures. The enzymatic complex was partially characterised for use in the production of glucose syrup. Type II wheat flour proved better than cassava bagasse as sole carbon source for amylase production. The optimum fermentation condition for both microorganisms was 96 hours at 30°C and the amylase thus produced was used for starch hydrolysis. The product of the enzymatic hydrolysis indicated that the enzyme obtained was glucoamylase, only glucose as final product was attained for both microorganisms. R. oligosporus was of greater interest than R. oryzae for amylase production, taking into account enzyme activity, cultivation time, thermal stability and pH range. Glucose syrup was produced using concentrated enzyme and 100 g L^?1 starch in a 4 hours reaction at 50°C. The bioprocess studied can contribute to fungus glucoamylase production and application.     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

A Split-Flow Enzyme Thermistor

Abstract

The split-flow system is comprised of two identical micro-columns, one of which contains an immobilized enzyme preparation, the other an inert support material.

The heat produced in each column on introduction of a sample is measured with thermistors placed in these columns. The use of a reference column virtually eliminates the influence on the measurements of artifactual signals as unspecific heat, i.e., heat not produced by the enzymic reaction. The performance of the split-flow enzyme thermistor at a variety of pH's, ionic strengths or viscosities associated with the sample has been investigated and compared with previously described alternative enzyme thermistor arrangements. In this comparative study glucose at a concentration of 5 · 10^?4 M was used throughout. On passage through the imnobilized glucose oxidase preparation this solution gave rise to a heat change At of about 0.01°C. The insensitivity of the system described herein towards such variations makes it particularly suitable for the analysis of metabolities present in crude solutions such as urine and skim-milk.     

Four-channel enzyme thermistor system for process monitoring and control in biotechnology

A newly developed four-channel enzyme thermistor system is presented and its application to biotechnological process monitoring is discussed. Different sugars were detected on-line during the cultivation of Cephalosporium acremonium and Bacillus licheniformis on technical media and during a starch hydrolysis process with immobilized thermostable enzymes. Immobilized enzymes and entrapped microorganisms were used as biological compounds in this biosensor.     

Use of Mesophilic Fungal Amylases Produced by Solid-state Fermentation in the Cold Hydrolysis of Raw Babassu Cake Starch

Amylases constitute one of the most important groups of industrial enzymes, presenting several applications, such as in the food, textile, and ethanol manufacturing. In this work, a starchy residue from the Brazilian agroindustry, namely babassu cake, was used for the production of amylases by solid-state fermentation and for obtaining sugar hydrolysates, which can be used as building blocks for future bioconversions. Eight filamentous fungi from the genera Aspergillus and Penicillium were screened. Regarding amylase production, A. awamori strains showed well-balanced endoamylase and exoamylase activities, while A. wentii produced an amylolytic complex much richer in the endo-acting enzymes. Simultaneous liquefaction and saccharification using the crude enzyme extracts from the four most promising fungal strains was then investigated applying DOE techniques. The highest total reducing sugar content (24.70 g L^?1) was obtained by the crude extract from A. awamori IOC-3914, corresponding to a hydrolysis yield of 52% of total starch in the cake, while A. awamori IOC-3915 produced the most appropriate extract in terms of glucose release (maximum of 5.52 g L^?1). Multivariate analysis of the DOE studies indicated that these extracts showed their best performance at 50–57 °C under acid conditions (pH 3.6–4.5), but were also able to act satisfactorily under milder conditions (36 °C and pH 5.0), when TRS and glucose released were about 65% of the maximum values obtained. These data confirm the high potential of the enzyme extracts under study for cold hydrolysis of starch.     

Flow injection analysis for glucose and urea with enzyme reactors and on-line dialysis

A flow injection system for glucose and urea determination is described. The glucose determination uses immobilized glucose oxidase in a reactor designed to give 100% substrate conversion. The hydrogen peroxide formed is converted to a coloured complex with 4-aminophenazone and N,N-dimethylaniline. The coupling is catalysed by a reactor containing immobilized peroxidase. The coloured complex is measured in a flow-through spectrophotometric cell. Urea is converted to ammonia in a reactor with immobilized urease and detected with an ammonia gas membrane electrode. Proteins and other interfering species from serum samples are removed in an on-line dialyzer. Calibration curves are linear for glucose in the range 1.6 × 10^-4–1.6 × 10^-2 M and for urea in the range 10^-4–10^-1 M. The samples are 25 μl for glucose determination and 100 μl for urea determination. Linear ranges can be changed by varying the sample sizes. The effects of the dialyser, enzyme reactors and detectors on dispersion are evaluated.     

Amperometric enzyme electrodes: Part II. Conducting salts as electrode materials for the oxidation of glucose oxidase

Results are reported for the direct oxidation of the enzyme glucose oxidase on 7 different one-dimensional conducting donor acceptor salts. Experiments conducted with the enzyme in bulk solution are shown to be in good agreement with theory. Three salts, made of the cations tetrathiafulvalinium (TTF⁺), N-methylphenazinium or quinolinium with the anion tetracyanoquinodimethanide (TCNQ^?) had the lowest background currents and were used to make membrane sensors for glucose. Analysis of the variation of current with glucose concentration identified the rate limiting processes as transport of gluycose through the membrane and electrochemical kinetics under unsaturated and saturated conditions respectively. The electrochemical rate constants for these three materials were all greater than 10^?2 cm s^?1. TTF⁺TCNQ^? is the material of choice and linear calibration plots were obtained for glucose concentrations between 50 μmol dm^?3 and 10 mmol dm^?3.     

Oxygen electrode-based enzyme immunoassay for the amperometric determination of hepatitis B surface antigen

Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1–100 μg 1^?1. A microcomputer is used for data acquisition and processing.     

Mn(II)-sodium dodecyl sulphate complex mimic enzyme-catalyzed fluorescence quenching of Pyronine B by hydrogen peroxide

Mn(II)-sodium dodecyl sulphate complex (Mn(II)-SDS) is used to mimic the active group of peroxidase. The catalytic characteristic of this mimic enzyme catalyst in the oxidation reaction of fluorescence substrate, tetraethyldiaminoxanthyl chloride (Pyronine B (PB)), with hydrogen peroxide has been studied. The experimental results show that Mn(II)-SDS complex has similar catalytic activity that of peroxidase. The steady-state catalytic rate depends upon mimic enzyme and substrate concentrations, and the Michaelis-Menten parameters K_m, V_max and K_cat are 7.6×10⁻⁶ M, 7.9×10⁻⁷ M s⁻¹ and 7.9 s⁻¹, respectively. The catalytic activity of Mn(II)-SDS complex is compared with those of HRP and Hemin. Though the catalytic activity of Mn(II)-SDS complex is 15.9% of that of HRP, it can catalyze the oxidation reaction of PB with hydrogen peroxide lead to fluorescence quenching of PB. Under optimum conditions, linear relationship between fluorescence quenching F₀/F and concentration of H₂O₂ is in the range of (0.0-3.6) × 10⁻⁷ M. The detection limit is determined to be 3.0×10⁻⁹ M. By coupling this mimic catalytic reaction with the catalytic reaction of glucose oxidase (GOD), glucose can be detected. Linear relationship between F₀/F and concentration of glucose is in the range of (0.0-1.4) × 10⁻⁷ M. The detection limit is determined to be 4.2×10⁻⁹ M. This method is applied to the determination of glucose in human serum and the results are in good agreement with the phenol-4-aminoantipyrine (4-AAP).     

Determination of Glucose in Diluted Blood with a Thermal Flow Injection Analysis Biosensor

Abstract

The glucose concentration in diluted whole blood has been measured, using a miniaturized thermal biosensor based on the enzyme thermistor principle. The biosensor is a small flow injection system. A sample volume of 20μl is injected into a flow of 50μl/min. The heat produced when the sample passes the enzyme column is measured with a thermistor connected to a Wheatstone bridge. The enzyme column contains glucose oxidase and catalase co-immobilized on a solid support material. Samples of whole blood usually cause problems in flow-systems. The blood cells tend to block the enzyme column and the back pressure increases. We have tested a superporous agarose material as enzyme support material using tenfold diluted samples of whole human blood. The blood was collected from the finger-tip and diluted with buffer containing an anticoagulant and sodium fluoride. The number of samples possible to inject and the accuracy compared to the Boehringer Mannheim Reflolux have been determined. At least 100 ten-fold diluted blood samples could be injected on a micro-column of superporous agarose. The obtained glucose concentration correlated well with the one obtained with the reference instrument.     

Studies of the kinetics of the interaction between N-trifluoroacetlyl-D-tryptophan and α-chymotrypsin by pulsed nuclear magnetic resonance

The Carr-Purcell experiment first used by Allerhand and Gutowsky for the determination of chemical exchange rates has been applied to the study of an enzyme inhibitor complex. Chemical shift and relaxation time data obtained by analysis of pulsed fluorine NMR data collected at 51 MHz are shown to be consistent with high resolution results assembled at 94° 1 MHz. The rate constants for dissociation of the N-trifluoroacetyl-D -tryptophan-α-chymotrypsin complex were determined to be 1 × 10⁴ s^?1 at 26°C and 2 × 10³ s^?1 at 6·5°C. The resonance position of the fluorine nuclei of the inhibitor is shifted downfield ～1 ppm upon complexation to the enzyme, and the trifluoromethyl group suffers some restriction of molecular motion in the bound state as indicated by T₁ and T₂ data.     

Potentially Antihyperglycemic from Biomass and Phycocyanin of Spirulina Fusiformis Voronikhin by in Vivo Test

Spirulina is one of the microalgae containing nutrients used as functional food and in theraupetic (active compound). Bioactive compound in Spirulina has antihyperglycemic activity or antidiabetes activity. Diabetes mellitus is a major health problem in the world. The research about the potential of microalgae as antihyperglicemic is important to do. The cultivation of microalgae was conducted in laboratory using flask and aquarium completed with non-stop aeration. Measurement of the antihyperglycemic activity in-vivo using mices that were fed with biomass containing Spirulina fusiformis Voronikhin and phycocyanin. Blood glucose levels were measured by methods of oral glucose tolerance test after a fasting period of 18 h. Oral administration of biomass and phycocyanin of Spirulina fusiformis V. at 0.15 mg · g^–1 and 0.30 mg · g^–1 proved to decrease the blood glucose level.     

On-line flow-injection monitoring of the enzyme inductor l-phenylalanine in the continuous cultivation of rhodococcus sp. M4

The o-phthaldialdehyde (OPA) assay for amino acids was adapted for flow injection analysis. With a dual-channel manifold and 2-μl injections, injection rates of 90–100 h^?1 were possible. Depending on the selected fluorimeter sensitivity, linear response ranges of 0.1–1.2 mM or 1–30 mM were obtained for l-phenylalanine with a relative standard deviation (RSD) of 0.9% for the slope of the calibration line. For ten injections of a 0.1 mM standard, the RSD was 1.6%. The detection limit was 0.01 mM. When the flow-injection method was combined with a fermentation sampling device and controlled by a timer, it was possible to monitor the decrease of l-phenylalanine on-line over a period of 87 h in the initial phase of a continuous cultivation of Rhodococcus sp. M4 producing the intracellular enzyme l-phenylalanine dehydrogenase. Correlation of the results with those obtained with an amino acid analyzer was good. Because OPA reacts with all primary amino groups, the applicability of the proposed method is restricted to cases in which only one amino acid and only small amounts of other amino group donators are present.     

In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen

The aim of this work was to optimize the conditions for in vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula × tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca²⁺, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca²⁺, 8 mM Mg²⁺, and 20 mM cellobiose. The in vitroβ-glucans were identified by hydrolysis of radioactive products, using specific enzymes. ¹³C-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1→3)-β-D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.     

Heavy metal ions in wines: meta-analysis of target hazard quotients reveal health risks

Background

Although the necessity of divalent magnesium and manganese for full activity of sugar nucleotidyltransferases and glycosyltransferases is well known, the role of these metal cations in binding the substrates (uridine 5'-triphosphate, glucose-1-phosphate, N-acetylglucosamine-1-phosphate, and uridine 5'-diphosphate glucose), products (uridine 5'-diphosphate glucose, uridine 5'-diphosphate N-acetylglucosamine, pyrophosphate, and uridine 5'-diphosphate), and/or enzyme is not clearly understood.

Results

Using isothermal titration calorimetry we have studied the binding relationship between the divalent metals, magnesium and manganese, and uridine 5'-phosphates to determine the role these metals play in carbohydrate biosynthesis. It was determined from the isothermal titration calorimetry (ITC) data that Mg⁺² and Mn⁺² are most tightly bound to PP _i , K_b = 41,000 ± 2000 M^-1 and 28,000 ± 50,000 M^-1 respectively, and UTP, K_b = 14,300 ± 700 M^-1 and 13,000 ± 2,000 M^-1 respectively.

Conclusion

Our results indicate that the formal charge state of the phosphate containing substrates determine the binding strength. Divalent metal cations magnesium and manganese showed similar trends in binding to the sugar substrates. Enthalpy of binding values were all determined to be endothermic except for the PP _i case. In addition, entropy of binding values were all found to be positive. From this data, we discuss the role of magnesium and manganese in both sugar nucleotidyltransferase and glycosyltransferase reactions, the differences in metal-bound substrates expected under normal physiological metal concentrations and those of hypomagnesaemia, and the implications for drug design.     

Measurement of biological parameters during fermentation processes

Efficient fermentation control requires the measurement of biological parameters. Three techniques were tested for monitoring. In the first, the NADH-fluorescence of micro-organisms was measured in batch and in continuous cultures under aerobic and anaerobic conditions, providing information on the metabolic status of the cells. The effects of cell concentration and of different substrates (glucose, ethanol and oxygen) were studied. The second technique is the calorimetric determination of various substrates, such as penicillin or enzymes, by an enzyme/thermistor device. With immobilized penicillin acylase (E.C. 3.5.1.11) or penicillinase (E.C. 3.5.2.6), penicillin was determined selectively in a fermentation broth. The thermistor was also used to measure penicillin acylase activity. The third technique is laser flow cytometry. A commercial double-beam flow cytometry system was used to determine cell size, light scattering and the protein, DNA and RNA contents of single cells. Flow cytometry allows rapid and sensitive control of fermentation processes with genetically modified E. coli 5K (pHM12) cells. The results of monitoring the cell size, light scattering, and protein and DNA contents of different micro-organisms during fermentation are outlined.     

The biosynthesis of the antibiotic obafluorin from p-aminophenylalanine in Pseudomonas fluorescens

The separate units which are used to construct the unique β-lactone antibiotic obafluorin (1) in Pseudomonas fluorescens are defined by the results of [U-¹³C]glucose incorporation. A key intermediate in the biosynthesis of (1) is established to be L-p-aminophenylalanine (7); L-p-nitrophenylalanine (8) is a relatively insignificant precursor. Similar results were obtained for p-nitrophenylacetic acid (9) which is also a metabolite of Ps. fluorescens. L-phenylalanine is an insignificant precursor for obafluorin (1).     

Modification of a Capillary for Electrophoresis by Electrostatic Self‐Assembly of an Enzyme for Selective Determination of the Enzyme Substrate

The integration of a separation capillary for capillary electrophoresis (CE) with an on‐column enzyme reaction for selective determination of the enzyme substrate is described. Enzyme immobilization is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the GOx produces hydrogen peroxide which migrates the length of the capillary and is detected amperometrically at the capillary outlet. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre‐ or post‐separation chemical modification of the analyte. The enzyme reactor is found to have an optimal response to glucose when a 5 : 1 mixture of PSS:GOx is used. Under these conditions the limit of detection for glucose is found to be between 5.0×10^?4 and 1.3×10^?3 M, dependent upon the inner‐diameter of the capillary. The apparent Michaelis‐Menten constant for the enzyme reaction was determined to be 0.047 (±0.001) M and 0.0037 (±0.0007) M for a 50 and 10 μm inner‐diameter capillaries, respectively. These results indicate that the enzyme reaction is efficient, having enzyme kinetics similar to that of a reaction occurring in solution. This enzyme immobilization method was also applied to another enzyme, glutamate oxidase, yielding similar results.     

Bioconversion of cellulose and simultaneous production of thermoactive exo- and endoglucanases by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Saccharification of cellulose is a promising method for production of biofuels. However, low bioconversion efficiency of cellulose to soluble sugars is a major challenge. In this study, a cellulolytic strain of Fusarium oxysporum was cultivated on pure cellulosic substrates (avicel, α-cellulose, carboxymethylcellulose and methylcellulose) and conversion efficiency into glucose was investigated. Production of exo- and endoglucanases during the bioconversion process was evaluated. Influence of pH on saccharification of cellulose and enzyme production by F. oxysporum were determined. Highest yield of glucose (1.76 μmol/ml) was obtained from F. oxysporum on methyl cellulose at 192 h under basal conditions. Liberated glucose under optimized condition of pH 6.0 at 96 h of fermentation was 2.12 μmol/ml with maximum production of exo- and endoglucanases (23.70 and 34.72 U/mg protein, respectively). The crude exo- and endoglucanases had optimum activities at pH 8.0, 70 °C and pH 7.0, 50 °C, respectively. The enzymes were stable over pH of 4.0–7.0 with relative residual activity above 60% after 1 h incubation. Exoglucanase activity was enhanced by Ca²⁺ and Cu²⁺ at 5 mM and Mg²⁺ at 10 mM. Endoglucanase activity was greatly enhanced in the presence of Mn²⁺, Ca²⁺, Mg²⁺, Cu²⁺ and Fe³⁺ at 5 and 10 mM. Activities of both enzymes were inhibited in the presence of Hg²⁺ at 5 and 10 mM. Results show that F. oxysporum possessed good cellulolytic enzyme system for efficient conversion of cellulose. Exhibited thermotolerance of exoglucanase with the striking tolerance of endoglucanase to metal ions demonstrate potentials of enzymes for biofuel industry.     

Fabrication of a Highly Sensitive Glucose Biosensor Based on Immobilization of Osmium Complex and Glucose Oxidase onto Carbon Nanotubes Modified Electrode

In this research a novel osmium complex was used as electrocatalyst for electroreduction of oxygen and H₂O₂ in physiological pH solutions. Electroless deposition at a short period of time (60 s), was used for strong and irreversible adsorption of 1,4,8,12‐tetraazacyclotetradecane osmium(III) chloride (Os(III)LCl₂) ClO₄ onto single‐walled carbon nanotubes (SWCNTs) modified GC electrode. The modified electrode shows a pair of well defined and reversible redox couple, Os(IV)/Os(III) at wide pH range (1–8). The glucose biosensor was fabricated by covering a thin film of glucose oxidase onto CNTs/Os‐complex modified electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The fabricated biosensor shows high sensitivity, 826.3 nA μM^?1cm^?2, low detection limit, 56 nM, fast response time <3 s and wide calibration range 1.0 μM–1.0 mM. The biosensor has been successfully applied to determination of glucose in human plasma. Because of relative low applied potential, the interference from electroactive existing species was minimized, which improved the selectivity of the biosensor. The apparent Michaelis‐Menten constant of GOx on the nanocomposite, 0.91 mM, exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this glucose biosensor.