Biochemical Engineering Journal (v.23, #3)


BEJ Keywords (IV).

Copolymeric beads consisting of cross-linked alginate–guar gum were found to fluidize well. The terminal velocity and bed expansion index were found to be 1339.4 cm h−1 and 5.9, respectively. The calculated particle Reynold number of 400 indicated laminar flow. The residence time distribution curves with bovine serum albumin showed intermediate dispersion behaviour according to axial dispersion model. The presence of guar gum in the beads led to binding of jacalin, with maximum binding capacity of 35 × 104  U ml−1 beads. The dynamic binding capacity at 5% breakthrough reflected efficient loading of the lectin. The haemagglutinating activity could be recovered at the level of 88% and the preparation was found to be 50-fold purified. SDS-PAGE showed a single band for the purified preparation.
Keywords: Alginate; Expanded bed affinity chromatography; Guar gum; Jacalin; Lectin; Protein purification;

The esterification of fatty acids of different chain lengths with isoamyl alcohol was studied in solvent free conditions and in supercritical carbon dioxide (SCCO2). Three different commercial lipases, Hog pancreas lipase (HPL), Lipolase 100T, and Novozym 435 were used for the synthesis of the esters. The results indicate that Novozym gave high conversion for the esterification of all acids, while HPL and Lipolase favored the conversion of higher and lower chain lengths of fatty acids, respectively.
Keywords: Biocatalysis; Enzymes; Lipase; Supercritical; Flavors;

Penicillin G acylase (PGA) is an important enzyme in β-lactam antibiotics pharmaceutical industry for the production of 7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) from cephalosporin G (Ceph-G) as well as 6-aminopenicillanic acid (6-APA) from penicillin G (Pen-G). In this work, immobilized PGA was utilized to catalyze the conversion of Ceph-G to 7-ADCA. Higher concentration of Ceph-G was found to show inhibition effect on the reaction. Both 7-ADCA and PAA (phenylacetic acid) were found to inhibit the activity of PGA, however, via different mechanisms. In this work, with low concentrations of Ceph-G substrate, the inhibited kinetic models were obtained by adding different concentrations of PAA or 7-ADCA. The influence from both products was discussed and compared. Both inhibition kinetic mechanisms were different in that PAA showed noncompetitive inhibition to PGA whereas 7-ADCA appeared to be of competitive inhibition.
Keywords: Immobilized penicillin G acylase; Cephalosporin G; 7-ADCA; PAA, inhibition kinetics, noncompetitive inhibition, competitive inhibition;

A mathematical model for the enzymatic biotransformation of benzaldehyde and pyruvate to R-phenylacetylcarbinol (PAC) and its associated by-products has been developed using a schematic method devised by King and Altman [E.L. King, C. Altman, A schematic method of deriving the rate laws for enzyme catalysed reactions, J. Phys. Chem. 60 (1956) 1375–1378] for deriving the rate equations for a complex enzyme-catalysed reaction. PAC is the commercial intermediate for the production of ephedrine and pseudoephedrine. A combinatorial theorem was applied using Visual Basic to create all of the possible reaction patterns for a simplified form of the pyruvate decarboxylase (PDC) biotransformation mechanism. The rate equations for substrates, product, and by-products have been derived from the patterns for yeast PDC and combined with a deactivation model for PDC from Candida utilis. The batch biotransformation profile generated by the model validated previously for a data set at initial substrate concentrations 50–150 mM benzaldehyde and 60–180 mM pyruvate, provided an acceptable fit for published data at initial concentrations of 400 mM benzaldehyde and 600 mM pyruvate.
Keywords: Biotransformations; (R)-phenylacetylcarbinol; Biokinetics; Enzyme biocatalysis; King and Altman procedure; Modelling;

Evaluation of a pH-stat feeding strategy on the production and recovery of Fab’ fragments from E. coli by Roeb García-Arrazola; Sun Chau Siu; Gerard Chan; Ian Buchanan; Billy Doyle; Nigel Titchener-Hooker; Frank Baganz (221-230).
A comparison between two feeding strategies in a gas blending fermentation process to produce Fab’ by Escherichia coli was carried out at 20 L scale. A pH-stat feeding strategy was proposed to address the issue of glycerol oscillations that occur in repeated batch fermentations. This feeding strategy was based on direct coupling of growth and concomitant production of H+ and this led to a constant minimum concentration of glycerol (main C-source) in the induction period. This change of environment resulted in a two-fold increase in the production of Fab’ compared to those obtained using a repeated batch strategy. A glycerol yield coefficient (Y X/glycerol in g cell/g glycerol) and product yield on biomass (Y P/X in mg product/g cell) of 0.39 and 5.91, respectively, were estimated for the pH-stat feeding fermentation. By contrast, Y X/glycerol and Y P/X were found to be 13 and 45% lower, respectively, for the repeated batch process. Therefore, considerable improvements in glycerol utilisation and product formation have been obtained using the pH-stat feeding strategy. Product localisation in the cell periplasm of >90% was obtained in both processes and a difference of product loss in the centrifugation step of less than 1% was observed. Overall, a method to improve production of Fab’ fragments without compromising primary recovery of cells by maintaining constant low levels of glycerol has been developed.
Keywords: Fab’ fragments; pH-stat; E. coli; Fermentation; Glycerol;

Polysaccharide production of Neisseria meningitidis (Serogroup C) in batch and fed-batch cultivations by Júlia Baruque-Ramos; Haroldo Hiss; Luciana Juncioni de Arauz; Rose Leila Mota; Maria Esther Ricci-Silva; Marcelo Fossa da Paz; Marta Massako Tanizaki; Isaías Raw (231-240).
Serogroup C polysaccharide from Neisseria. meningitidis constitutes the antigen for the vaccine against the disease caused by this bacterium. Aiming at enhancing the final polysaccharide concentration as well as the overall yield factor (polysaccharide/biomass), 20 cultivations were carried out in Frantz medium in a 13 L bioreactor at 35 °C, 0.5 atm, 400 rpm and air flowrate of 2 L/min. A series of nine batch experiments was carried out under three different conditions (with control of dissolved oxygen at 10%, with control of pH at 6.5 and without dissolved oxygen and pH controls). Another set of runs consisted of 11 fed-batch cultivations without dissolved oxygen control, varying glucose concentration from less than 1.0–3.0 g/L, four of which performed controlling the pH at 6.5, and four under partial fed-batch conditions. The highest polysaccharide concentration (0.26 g/L) and the overall yield (0.16 g/g), were obtained in batch and partial fed-batch experiments when glucose concentration was maintained below 1.0 g/L. An empirical relation is proposed to relate the specific production rate of polysaccharide to glucose concentration during the stationary growth phase of the fed-batch runs. The obtained polysaccharide satisfies the molecular weight criterion, being a suitable antigen for vaccine production.
Keywords: Neisseria meningitidis; N-Acetylneuraminic acid; Batch processing; Fed-batch culture; Growth kinetics; Production kinetics;

Metabolic mechanisms involved in hydroxylation reactions of diphenyl compounds by the lignin-degrading basidiomycete Phanerochaete chrysosporium by Nobuhiro Hiratsuka; Masafumi Oyadomari; Hiroaki Shinohara; Hiroo Tanaka; Hiroyuki Wariishi (241-246).
Biphenyl (BP), biphenylene (BN), dibenzofuran (DF), dibenzo-p-dioxin (DD), and diphenyl ether (DE) were utilized as diphenyl substrates for the lignin-degrading basidiomycete, Phanerochaete chrysosporium and its extracellular enzyme, lignin peroxidase (LiP). Among these compounds, only BN and DD were oxidized by LiP. Cyclic voltammetry measurement revealed that BN and DD possess lower redox potentials than other diphenyl substrates utilized, being accordance with a reactivity of LiP. Although the degradations of BP, DF and DE were not extracellularly initiated by LiP, they were metabolized via an intermediate formation of hydroxylated products by intracellular enzymes. Upon addition of piperonyl butoxide, a cytochrome P450 inhibitor, these hydroxylation reactions were effectively inhibited, indicating the involvement of cytochrome P450s. Thus, two individual mechanisms seem to be compulsory for P. chrysosporium to metabolize a wide variety of recalcitrant diphenyl compounds; one is the activation of the aromatic ring via LiP catalyzed one-electron oxidation forming the aryl cation radical and the other is the activation of molecular oxygen by cytochrome P450 enzymes causing hydroxylation reactions on the aromatic ring.
Keywords: Biodegradation; Bioremediation; Enzyme activity; Diphenyl compound; Filamentous fungi; Oxidation reactions;

The energy efficiency of the cell disruption process for the recovery of intracellular proteins depends on the physical strength of the cell wall of the microorganism and on the intracellular location of the target enzymes, also the way in which the stress is applied (effectiveness). Heat stress was found to induce translocation of the target enzyme (β-galactosidase) and also to promote aggregation of the total protein leached out in the suspension after their translocation. Translocation provides an external means to reduce the severity of the cell disruption process and reduction in the energy requirements for the same. This aggregated protein could be removed by centrifugation prior to cell disruption. Thus, the purity and selectivity of the target enzyme could be substantially improved, along with a reduction in the energy required for disruption, by subjecting cells to heat stress. The kinetics of translocation are reported and depend on variation in location factor (LF), possibly enabling the heat treatment protocol to be optimized.
Keywords: Cell disruption; Cavitation; Translocation; Location factor; Enzymes; Intracellular proteins;

Adsorption of BSA onto sulfonated microspheres by Jie Hu; Songjun Li; Bailing Liu (259-263).
The adsorption of bovine serum albumin (BSA) onto modified PMMA microspheres was studied. The sulfonate groups on the surface of microspheres play an important role in this system. From microsphere 1 to 4, with the increase in amount of sulfonate groups on the surface of microspheres, the apparent constants increased from 2.28 to 3.18 × 10−2  min−1. The adsorption isotherms were fitted to the Langmuir–Freundlich isotherm. With the increase of sulfonate groups on the surface of microspheres, C m values increased and K values decreased. Close to the isoelectric point of BSA, the adsorption amount exhibits a maximum. A higher or lower pH results in the significant decrease of the adsorption amount. And with the increase of ionic strength, the adsorption of BSA onto microspheres decreased. This may be responsible for the binding of Cl and BSA, which resulted in a degree of change in the conformation and local charge of BSA macromolecules.
Keywords: BSA; Sulfonate group; Adsorption; Microspheres;

The biochemical kinetic of simultaneous saccharification and fermentation (SSF) for lactic acid production by fungal species of Rhizopus arrhizus 36017 and Rhizopus oryzae 2062 was studied with respect to growth pH, temperature and substrate. Both R. arrhizus 36017 and R. oryzae 2062 had a capacity to carry out a single stage SSF process for lactic acid production from potato starch wastewater. The kinetic characteristics, termed as starch hydrolysis, accumulation of reducing sugars, lactic acid production and fungal biomass formation, were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 °C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.85–0.92 g/g associated with 1.5–3.5 g/l fungal biomass produced in 36–48 h fermentation. R. arrhizus 36017 had a higher capacity to produce lactic acid, while R. oryzae 2062 produced more fungal biomass under similar conditions.
Keywords: Fungal biomass; Lactic acid; Reducing sugars; Rhizopus arrhizus; Rhizopus oryzae; Simultaneous saccharification and fermentation; Starch hydrolysis;

Optimal aeration control of industrial alternating activated sludge plants by B. Chachuat; N. Roche; M.A. Latifi (277-289).
The operation of wastewater treatment plants is challenging for many small communities for economical and technical reasons. In this work, optimal control of the aeration system is considered to improve the efficiency and reliability of the activated sludge process, with application to an industrial alternating activated sludge (AAS) treatment plant. Two problems are formulated: the first one deals with the minimization of nitrogen discharge and the second one addresses the minimization of the energy consumption. In either cases, special emphasis is placed on the long-term behavior of the treatment plant in order to ensure that the optimal aeration strategies are safe and durable from the process point of view. Their solutions show that significant improvements can be achieved with respect to the current aeration strategy, with reductions up to 37% for the first problem and around 27% for the second one. Large performance improvements are also obtained when comparing the optimal aeration strategies to a conventional oxygen-based controller.
Keywords: Wastewater treatment; Alternating activated sludge process; Dynamic modeling; Aeration control; Optimization;