E. H. M. Melo

Lipase from a Brazilian strain of Penicillium citrinum

A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain ofPenicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40–60% fraction. The optimum temperature for enzyme activity was found in the range of 34–37°C. However, after 30 min at 60°C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28°C) for 8 mo. This result in lipase stability suggests an application of lipases fromP. citrinum in detergents and other products that require a high stability at room temperature.

Glucose Biosensor Using Glucose Oxidase Immobilized in Polyaniline

A biosensor for glucose utilizing kinetics of glucose oxidase (EC 1.1.3.4.) was developed. The enzyme was immobilized on polyaniline by covalent bonding, using glutaraldehyde as a bifunctional agent. The system showed a linear response up to 2.2 mM of glucose with a response time of 2.5–4.0 min. In addition, the immobilized enzyme had a higher activity between pH 6.5 and 7.5. The system retained 50% of its activity after 30 d of daily use. The optical absorption spectra of the polyaniline/glucose oxidase electrode after glucose had been added to the buffer solution showed that the absorption band around 800 nm had changed considerably when glucose was allowed to react with the electrode. This optical variation makes polyaniline a very promising polymer for use as a support in optical sensor for clinical application.

Lipase production by a Brazilian strain of Penicillium citrinum using an industrial residue

Penicillium citrinum produced a lipase activity (5786 U/l), on a vegetable oil refinery residue as carbon source, higher than the one (2630 U/l) obtained on olive oil. Lipase activity was measured with triolein as substrate and the free fatty acids released were estimated. Ammonium chloride (0.75%) was the best nitrogen source (6738 U/l) compared to ammonium sulfate (5670 U/l), and urea caused a decrease of 85% in this lipase production (898 U/l).

Production of extracellular lipase by the phytopathogenic fungus Fusarium solani FS1

A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v) peptone plus 0.5% (v/v) olive oil. Glucose (1% w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v) resulted in a reduced lipase production while increased olive oil concentration (above 0.5%) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30oC and a good enzyme stability (80% activity retention) was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50oC. Lipase activity was stimulated by the addition of n-hexane to the culture medium supernatants, in contrast to incubation with water-soluble solvents.

Immobilization of Candida rugosa lipase on Magnetized Dacron: Kinetic Study

Candida rugosa lipase has been covalently immobilized on ferromagnetic azide polyethyleneterepthalate (Dacron) with specific activity retention of 16% for 4-nitrophenyl palmitate and 24% for hydrolysis of triolein in hexane. The immobilized enzyme was more thermal stable than the soluble one, retaining 78.8% of the activity after 1 h at 60°C. Also, this immobilized derivative was stable at the storage at 4°C. It has been used 5 cycles for pNPP hydrolysis without loss of activity. Soluble and immobilized Candida rugosa lipase showed a Michaelian behavior for fatty acid 4-nitrophenyl esters and different apparent KM values: 0.110 mM and 0.124 mM (4-nitrophenyl palmitate – C16); 0.193 mM and 0.235 mM (4-nitrophenyl laurate – C12) and 0.206 mM and 0.119 mM (4-nitrophenyl butyrate – C4), respectively. The immobilized lipase was more efficient for catalyzing the hydrolysis of 4-nitrophenyl esters with short chain length fatty acid (4-NPB – C4) than soluble enzyme. The ferromagnetic Dacron-lipase derivative was able to catalyze the synthesis of triolein from glycerol and oleic acid with 50% of conversion after 72 h at 40°C.

Ascorbic acid determination in biological fluids using ascorbate oxidase immobilized on alkylamine glass beads in a flow injection potentiometric system

A flow injection method was developed aimed at the determination of ascorbic acid in biological fluids, particularly fruit juices. The enzyme ascorbic oxidoreductase (EC 1.10.3.3), extracted fromCucurbita maxima, was immobilized onto alkylamine glass beads using glutaral-dehyde as a bifunctional agent. The ascorbic acid concentration was related to oxygen saturation. Fall in oxygen concentration, as a result of ascorbic acid oxidation, was detected by a low cost, homemade oxygen electrode. The calibration graph was linear over the range 0.05 to 3.00 mM (RSD 1%), the maximum number of samples that could be analysed was 90/h. The immobilized enzyme retained its initial activity for 2 mo with more than 600 assays.

Immobilization of pneumococcal polysaccharide vaccine on silicon oxide wafer for an acoustical biosensor

One the most important aspects of a biosensor is related to immobilization and maintenance of specific reference compounds on sensing surfaces. A method for the immobilization of polysaccharides to a silicon oxide surface intended for Surface Acoustical Waves (SAW) sensors is described. Silicon oxide is a hydrophobic inorganic support used for the fabrication of many electronic devices. The pneumococcal polysaccharide (PPS) vaccine is immobilized via Protein A after pre-treatment of the surface with hydrochloric acid. The effects of non-specific binding are discussed. The results indicate that the immobilization of PPS via Protein A increases the sensitivity of detecting Streptococcus pneumoniae antibodies in human sera and offers greater reproducibility of response compared with ELISA methods. The principles of this technique are simple and are applicable to the immobilization of many capsular polysaccharides.

Simple and inexpensive Flow Injection Analysis for determination of sucrose using invertase and glucose oxidase immobilised on glass beads

A Flow Injection Analysis (FIA) for sucrose using invertase (E.C. 3.2.1.26), mutarotase (E.C.5.1.3.3) and glucose oxidase (E.C.1.1.3.4) was developed. The enzymes were immobilised on glass beads using glutaraldehyde. The sucrose concentration was related to oxygen saturation. Fall in O2 concentration, as a result of sucrose oxidation, was detected by a low cost, home-made O2 electrode. The system was able to measure sucrose from 0.025 to 100mM with a response time of 6min using 200 μl of sample, with an apparent Km of 42mM of sucrose. The system has been operated satisfactorily for 50 days without loss any initial activity.

Biological delignification of Brazilian lignocellulosic residues

ABSTRACT Phanerochaete chrysosporium has been grown on pieces of banana tree trunks. As a result of the semi-solid fermentation, using glucose as carbon source, it was found that the lignin peroxidase activity was 206 U/L on the fifth day of fermentation and 140 U/L on the tenth day. A reduction of 32% of the lignin content in the native banana tree trunk was observed after 15 days of fermentation. These results have shown that P. chrysosporium grown on banana tree trunks is a potential microorganism for an alternative process for cellulose and paper production with environmental benefits.

2.28 – Flow Injection Analysis (FIA) for Determination of Sucrose using GOD and Invertase Immobilised on Glass Beads

This chapter discusses a flow injection analysis (FIA) study for determination of sucrose using GOD and invertase immobilized on glass beads. The measurement of sucrose is important in the food and fermentation industries. It is thus very important to develop a fast, simple and reproducible method for determination of sucrose. The principle of this sensor is based on monitoring the decrease in dissolved oxygen resulting from three enzyme reactions in the presence of sucrose. In this study, invertase (E.C.3.2.1.26.) and mutarotase (E.C.5.1.3.3.) were obtained from bakers yeast ( Saccharomyces cerevisiae ); GOD (E.C.1.1.3.4.) was obtained. The enzymes were immobilized by covalent binding using glutaraldehyde as bifunctional agent on alkylamine glass beads. The flow system was used to evaluate the sensor under dynamic conditions. The working electrode was a home-made oxygen electrode. A sample of sucrose was injected into the system and carried by citrate–phosphate buffer at flow rate of 0.6 ml/min upward through the column containing the immobilized enzyme. An apparent Km of 0.048 M was found. This system shows a linear output from 2.5 to 100 mM of sucrose.