J. M. S. Cabral

Isolation of a β-Carotene Over-Producing Soil Bacterium, Sphingomonas sp.

A carotenoid-accumulating bacterium isolated from soil, identified as a Sphingomonas sp., grew at 0.18 h−1 and produced 1.7 mg carotenoids g−1 dry cell, among which β-carotene (29% of total carotenoids) and nostoxanthin (36%). A mutant strain, obtained by treatment with ethyl methanesulfonate, accumulated up to 3.5 mg carotenoids g−1 dry cell. Accumulation of β-carotene by this strain depended on the oxygenation of the growth medium, with maximal accumulation (89%) occurring under limiting conditions. β-Carotene accumulation could be further enhanced by incubating the cells in the presence of glycerol (either not or only slowly assimilated) and yeast extract resulting in an accumulation of 5.7 mg β-carotene g−1 dry cell wt. The strain used lactose as carbon source with similar biomass and carotenoid production, providing a viable alternative use for cheese whey ultra-filtrate.

Callus and suspension cultures for biomass production ofCynara cardunculus (Compositae)

SummaryFriable calli, obtained from hypocotyl and leaf segments ofCynara cardunculus, were used for the production of cell suspension cultures in liquid Gamborg B5 medium supplemented as for callus obtention. The low inoculum cell suspension cultures (6.9×104 cells.ml−1) presented a td=136.8 hours while those obtained with a high inoculum (15.6×104 cells.ml−1) reached a td=33.6 hours.

Thermostability of α-chymotrypsin encapsulated in reversed micelles

SummaryThe influence of water content, additives, pH and substrate concentration on the thermostability of α-chymotrypsin entrapped in a reversed micellar system of the cationic surfactant TTAB/heptane/ chloroform, was studied. Increasing the water level inside the reversed micelles diminishes the enzyme stability. Enzyme stability enhancement was achieved with the addition of glycerol, by increasing the nucleophile concentration or by decreasing the buffer pH.

Callus and suspension culture ofSilybum marianum. Biosynthesis of proteins with clotting activity

SummarySilybum marianum plants contain a proteinase with clotting activity which is used for the production of soft cheese. The influence of the concentrations of phytohormones (2,4-D and kinetin), of aminoacids and sucrose on the growth rate and intracellular protein content of calli and suspension cultures was studied. The clotting: proteolytic activity ratio was compared with that of a microbial rennet.

Thermostability of α-chymotrypsin in water/organic solvent systems

SummaryThe influence of pH, temperature, substrate concentration and organic solvents (dimethylformamide, dimethylsulfoxide) on the α-chymotrypsin stability in a water/organic solvent system was studied. The enzyme activity was measured as the dipeptide, AcPheLeuNH2 synthesis and the ester substrate hydrolysis. Enzyme stability was enhanced by lower pH and temperature values and higher substrate concentrations. Dimethylsulfoxide allowed an higher enzyme stability than dimethylformamide. α-Chymotrypsin displayed an higher stability in the water medium when it was compared to the organic system.

Scaling-up Ex Vivo Expansion of Mesenchymal Stem/Stromal Cells for Cellular Therapies

The significantly large cell doses required in clinical trials with mesenchymal stem/stromal cells (MSC) demand for an efficient production of clinical-scale cell numbers. However, traditional cell culture techniques present several limitations making them unsuitable for the production of large numbers of MSC. Moreover, monitoring and control of MSC expansion are critical to provide a safe and reliable cell product for clinical settings. Bioprocess engineering, in particular xadbioreactors, offers the adequate tools to develop and optimize an efficient, cost-effective, and easily scalable culture system for the large-scale expansion of human MSC for cellular therapy.

Peptide Synthesis by Microencapsulated Chymotrypsina

The need for analogues of biologically active small peptides such as hormones, neuropeptides, and immunogenes and the preparation of modified protein analogues for protein chemistry studies have led to the development of methods for peptide synthesis. The use of proteolytic enzymes as biocatalysts for the peptide bond formation has several advantages over the well-established chemical synthesis. Peptides are obtained more rapidly and in higher yields when enzymatic synthesis is accomplished in organic media. In this work, the dipeptide AcPheLeuNH, was synthesized from acetylphenylalanine and leucinamide using a-chymotrypsin microencapsulated in reverse micelles of tetradecyltrimethylammonium bromide in a heptane/chloroform (1 : 1) solution.

Effect of aeration on Cynara cardunculus plant cell cultures

The species Cynara cardunculus is traditionally used in Portugal to produce tasteful and valuable cheese.

Continuous Coagulation of Milk using Immobilized Cells of Cynara Cardunculus

Plant rennets are used in Portugal for the small-scale manufacture of certain varieties of cheese, namely “Serra” and “Serpa”. In the traditional preparation of these types of cheese the clotting stage is induced by immersing the flowers of the native Compositae Cynara cardunculus or Silybum marianum in the milk vats. However, the flowers are, by themselves, a potential source of microbial contamination of the product. This problem could be overcome by plant cell culture. Previous work demonstrated that in vitro cultivated cells of both species present clotting activity (Fevereiro et al. 1986, Esquivel et al. 1986), no clotting activity being found in the supernatant (Fonseca et al. 1987).

Milk Clotting with Free and Immobilized Plant Cells of Silybum marianuma

Proteinases with clotting activity are synthesized in vivo by two species of Compositae, Cynara cardunculus, and Silybum marianum. These cardoons grow spontaneously in Portugal, their dried flowers being traditionally used in the manufacture of two varieties of soft cheese, Serra and Serpa. This type of cheese presents relatively low shelf life, due to microbial contamination derived from the presence of the cardoon during the milk-clotting stage. The massive aseptic in vitro production of cardoon cells is a tentative solution to overcome the contamination problem in order to obtain a steady production of cheese of improved quality and with an extended shelf life. The f n vitro conditions for the establishment of callus and suspension cultures of s. marianum were described by us previously.’ The effect of phytohormones and sucrose concentration on the specific growth rate of the callus cultures was studied, as was the influence of the presence of amino acids on the growth and protein content of the calli. Conditions for achieving friable calli suitable for the initiation of suspension cultures were discussed. An association between the growth rate of suspended cells and their total intracellular protein content was observed.’ In the present work, the effects of storage, permeabilization, and immobilization on the proteolytic and clotting properties of S. marianum cells were studied. Azocasein (Sigma) and pasteurized milk were used as substrates for cardoon cells to test the proteolytic and clotting activities, respectively. The microbial rennet (“Rennilase 1 SOL”) was a gift from Novo Industri, Denmark. Sodium alginate (BDH, U.K.), K-carrageenan (“Gelcarin CIC”, from FMC), and agar (Iberagar, Portugal) were used as matrices for cell gel entrapment. The coarse pore polyurethane foam was a gift from Scotfoam, Pennsylvania, USA. The polyurethane prepolymer (“Hypo1 2002”) and the silica gel (0,l-0,5 mm particle size) were samples kindly supplied by Grace, F.R. Germany. The clotting activity was assayed in a shaking bath (80 strokes/min, 3OOC) using 30 ml pasteurized milk, pH 6.4, and 100 mg free or immobilized wet cell mass. The proteolytic activity was assayed at 3OoC through the action of 100 mg free or