Studies on improved techniques for immobilizing and stabilizing penicillin amidase associated with E. coli cells

Abstract: A novel biosensor based on immobilised whole cell Chlorella vulgaris microalgae as a bioreceptor and interdigitated conductometric electrodes as a transducer has been developed and tested for alkaline phosphatase activity (APA) analysis. These sensors were also used for the detection of toxic compounds, namely cadmium ions, in aquatic habitats. Algae were immobilised inside bovine serum albumin (BSA) membranes cross-linked with glutaraldehyde vapours. The detection of the local conductivity variations caused by algae enzymatic reactions could be achieved. The inhibition of C. vulgaris microalgae Alkaline phosphatase activities in presence of cadmium ions was measured. These results were compared with measurements in bioassays. It finally appeared that conductometric biosensors using algae seemed more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for the first experiments was 1 ppb of Cd2+). The main advantages of these alkaline phosphatase biosensors consist of their high specificity in regard to the toxic compounds they enable to detect, but also on their high stability since contrary to enzymatic biosensors, they use whole algae cells with APs on their walls.

Abstract: Scientific evidence in the prevention and treatment of various disorders is accumulating regarding probiotics. The health benefits supported by adequate clinical data include increased resistance to infectious disease, decreased duration of diarrhea, management of inflammatory bowel disease, reduction of serum cholesterol, prevention of allergy, modulation of cytokine gene expression, and suppression of carcinogen production. Recent ventures in metabolic engineering and heterologous protein expression have enhanced the enzymatic and immunomodulatory effects of probiotics and, with time, may allow more active intervention among critical care patients. In addition, a number of approaches are currently being explored, including the physical and chemical protection of cells, to increase probiotic viability and its health benefits. Traditional immobilization of probiotics in gel matrices, most notably calcium alginate and κ-carrageenan, has frequently been employed, with noted improvements in viability during freezing and storage. Conflicting reports exist, however, on the protection offered by immobilization from harsh physiologic environments. An alternative approach, microencapsulation in “artificial cells,” builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading, and greater control of parameters. This review summarizes the current clinical status of probiotics, examines the promises and challenges of current immobilization technologies, and presents the concept of artificial cells for effective delivery of therapeutic bacterial cells.

Abstract: Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. This paper, for the first time, discusses the use of lactic acid bacteria for the production of ferulic acid. Specifically, we have used Lactobacillus cells and microencapsulation so that ferulic acid can be produced continuously using various types of fermentation systems. Bacteria were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and the production of ferulic acid by lactobacilli was detected using a real-time high-performance liquid chromatography (HPLC)-based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.

Abstract: Whole cell based biosensor is prepared by immobilizing Chlorella sp. microbes over glassy carbon electrode. The proposed biosensor is optimized for characteristics like substrate concentration, pH, response time, and durability. The electrode responds linearly in concentration range of 10 −14 M to 10 −6 M for mercury and showed its rare selectivity over silver, alkali metals, alkaline earth metals and transition metals with an expected life of 14 days.

Abstract: A whole-cell environmental biosensor was fabricated on a diamond electrode. Unicellular microalgae Chlorella vulgaris was entrapped in the bovine serum albumin (BSA) membrane and immobilized directly onto the surface of a diamond electrode for heavy metal detection. We found that the unique surface properties of diamond reduce the electrode fouling problem commonly encountered with metal electrodes. The cell-based diamond biosensor can attain a detection limit of 0.1 ppb for Zn2+ and Cd2+, and exhibits higher detection sensitivity and stability compared to platinum electrodes.

Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.

Abstract: Immobilised penicillin acylases, both penicillin G and penicillin V acylases have been used extensively in the industrial production of 6-aminopenicillanic acid. This review presents an overview of the state-of-the art in the production of the enzyme and its application in the manufacture of 6-aminopenicillanic acid, the key intermediate in semi-synthetic penicillins production. Penicillin acylases used industrially are immobilised on solid matrices mostly by covalent bonding or by copolymerisation and such preparations of the enzymes are available commercially. Both stirred-tank and packed-column type of reactors have been used in the hydrolysis of penicillin

Abstract: Penicillin amidase was extracted from Escherichia coli ATCC 9637, grown on phenylacetic, acid and glutamate, and purified by fractional ion with streptomycin sulphate, ammonium sulphate and polyethylene glycol, followed by chromatography on DEAE–cellulose. The purification factor was 100–200 × and the overall yield was about 115%. The enzyme was chemically attached to derivatives of cellulose and the kinetics of these insolubilized penicillin amidase preparations was investigated.

Abstract: Mise au point d'un materiel immobilise a activite enzymatique conservee, utilisable pour la production industrielle de penicilline semi-synthetique. L'immobilisation de penicilline acylase sur un support type chitosane est decrite: differentes formes du support sont testees: poudre, particules ou billes de chitosane. Differentes methodes d'immobilisation sont egalement presentees: adsorption-reticulation, liaison covalente. Deux types de reacteur sont utilises suivant le type de support choisi: reacteur agite pour la poudre et les particules de chitosane, reacteur lit fixe pour les billes. L'influence de ces differents types d'immobilisation sur l'activite enzymatique de la penicilline G acylase est mesuree

Abstract: For continuous production of 6-aminopenicillanic acid (6-APA) the microbial cells ofEscherichia coli ATCC 9637 having high penicillin amidase (penicillin amidohydrolase, E.C. 3. 5. 1. 11) activity were immobilized by entrapment in a polyacrylamide gel lattice. Enzymatic properties of penicillin amidase of the immobilizedE. coli cells were investigated and compared with those of the intact cells. With regard to optimal pH and temperature, no marked difference was observed. The heat stability was somewhat increased by immobilization of the cells. The enzyme activity of the immobilized cell column was stable, and its half-life was 17 days at 40°C and 42 days at 30°C. From the effluent of the column, 6-APA was easily obtained in a good yield.