Biochemical Engineering Journal (v.16, #1)


BEJ Keywords (IV).

The synthesis of the biopolymer poly(3-hydroxy butyrate) P(3HB) was studied in a batch reactor using a mutant strain of Azotobacter vinelandii utilizing glucose. The objective of the present study was to optimize the cost of production by using glucose as substrate. In order to achieve this, a mutant strain was developed using a UV-mutagenic technique which is capable of utilizing glucose as substrate and giving a better yield. The effect of temperature, pH, glucose concentration and inoculum concentration on the production of P(3HB) using the optimized media was studied. The optimum temperature, pH, glucose concentration and inoculum concentration for growth and P(3HB) synthesis were found to be 30 °C, 7.5, 2.4% (w/v), and 0.63 g/l, respectively. The experiments were carried out to establish the kinetics of P(3HB) production by measuring cell mass, P(3HB) and glucose concentrations as a function of time in the batch reactor under optimum conditions. Growth kinetic models such as Monod, logistic and modified logistic models were used to predict the experimental data. The respective growth models incorporated in the Leudeking–Piret model and modified Leudeking–Piret models were used to predict P(3HB) formation rates and glucose consumption rates.
Keywords: Poly(3-hydroxy butyrate); Mutant strain Azotobacter vinelandii; Glucose; Batch kinetic modeling;

Sequential expression of recombinant proteins and their separate recovery from a Pichia pastoris cultivation by Jyh-Ming Wu; Lieng-Lung Chieng; Tsu-An Hsu; Cheng-Kang Lee (9-16).
Pichia pastoris strains with capability to sequentially express two recombinant proteins under control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GAP) and the methanol-induced alcohol oxidase (AOX1) promoters have been constructed. The intracellular β-galactosidase (β-gal), secreted human serum albumin (hSA) and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) are used as model proteins to demonstrate their sequential expression and separate recovery. The strain X33/ZCSF cultivated in YPD medium results in high-level constitutive expression of β-galactosidase activity and methanol-induced, secreted highly glycosylated hGM-CSF expression. The β-galactosidase activity and hGM-CSF are separately recovered from cells pellet and supernatant. On the other hand, the strain GS115/CSFA cultivated in YPD medium constitutively expresses secreted hGM-CSF along with cell growth. After a high cell concentration is achieved, the YPD medium is then replaced with YPM medium to induce the expression of the AOX1 promoter controlled hSA. The sequentially expressed hGM-CSF and hSA can be separately recovered from the spent YPD and YPM medium.
Keywords: Pichia pastoris; Sequential expression; GAP promoter; AOX1 promoter; hGM-CSF;

Synthesis of 6-O-unsaturated acyl l-ascorbates by immobilized lipase in acetone in the presence of molecular sieve by Ko Kuwabara; Yoshiyuki Watanabe; Shuji Adachi; Kazuhiro Nakanishi; Ryuichi Matsuno (17-22).
6-O-Unsaturated acyl l-ascorbates were synthesized through immobilized-lipase-catalyzed condensation of ascorbic acid and various unsaturated fatty acids. The equilibrium conversion was higher at a higher molar ratio of a fatty acid to ascorbic acid for the synthesis of every acyl l-ascorbate in the absence of a molecular sieve. For the synthesis of oleoyl l-ascorbate, the addition of molecular sieve 4 Å into the reaction system increased both the initial reaction rate and the conversion. However, the conversion gradually decreased on prolonged reaction time. When an excess amount of molecular sieve was added into the system, the conversion was lowered. Furthermore, the molecular sieve adsorbed and degraded the oleoyl ascorbate, and these rate processes were also analyzed.
Keywords: Lipase; Acyl l-ascorbate; Unsaturated fatty acid; Molecular sieve;

The kinetic parameters of fungi like Aspergillus awamori may vary significantly according to culture conditions, such as oxygen and fermentation media. The knowledge of maximum growth rate and growth yields is necessary for process optimisation, modelling and scale-up. The measurement of such parameters is hindered on fermentations where complex, heterogeneous medium is utilised. This study focuses on the determination of kinetic parameters such as maximum specific growth rate (0.28 h−1) and growth yields on substrate (0.31 g g−1) and oxygen (0.22 g g−1) for A. awamori growing under oxygen limiting conditions on whole wheat flour in submerged culture. The low growth yields indicated the effect of oxygen depletion on fungal kinetic parameters. A specific glucoamylase production of 72 U g−1  h−1 has been observed. The unstructured mathematical model used to simulate fermentation variables takes into account fungal growth, dissolved oxygen, glucose consumption, starch hydrolysis, glucoamylase activity, volumetric oxygen transfer coefficient and apparent viscosity. The fungal growth has been estimated using on-line measurements of CO2 evolution during fermentation.
Keywords: Submerged culture; Aspergillus awamori; Kinetic parameters; Modelling; Oxygen limitation;

Regioselective reduction of a steroid in a reversed micellar system with enzymatic NADH-regeneration by Hidehiko Hirakawa; Noriho Kamiya; Tsuyoshi Yata; Teruyuki Nagamune (35-40).
The regioselective reduction of androstandione to androsterone by 3α-hydroxysteroid dehydrogenase (HSDH) from Pseudomonas testosteroni was coupled to a cofactor (NADH) regeneration system with the oxidation of ethanol by yeast alcohol dehydrogenase (YADH) in sodium dioctyl sulfosuccinate (AOT)/isooctane reversed micelles. Each reaction was dominated by the system’s water content (W 0=[H2O]/[AOT]). The catalytic activity of YADH increased monotonically with increasing W 0, whereas HSDH showed bell-shaped dependency on W 0. Using a reversed micellar system increased stability of both enzymes in comparison with an aqueous system and prolonged NADH-regeneration; namely, a seven-fold increase in the total turnover number of NADH was achieved. These results suggest that the reversed micellar system is a promising choice for conjugate enzymatic reactions with dehydrogenases in a nonaqueous media.
Keywords: Bioconversion; Cofactor regeneration; Enzymes; Microemulsions; Reversed micelles; Dehydrogenase;

State-of-the-art: rheological characterisation of wastewater treatment sludge by Isabelle Seyssiecq; Jean-Henry Ferrasse; Nicolas Roche (41-56).
Since wastewater treatment has been a subject of interest, non-Newtonian flows, such as sludge flows, have been widely studied in the literature, most of the time from a rheological point of view. It is well known that the hydrodynamic behaviour of sludge flows is of prime importance to optimise process parameters of wastewater plants on one hand, as well as those of excess sludge retreating processes on the other hand. The present study is devoted to a synthesis of articles dealing with the rheological characterisation of biological wastewater treatment plant sludges (activated or concentrated ones) and some other concentrated suspensions such as microorganism ones. Attention is notably given to the rheological methods used and to rheological equations and behaviours observed in the different articles. Correlation proposed by some authors linking rheological properties to physico-chemical parameters of suspensions, or to some operating parameters of a process involving the use of sewage sludges are also given in this literature review.
Keywords: Biological wastewater treatment; Rheology; Sludge; Literature review;

Pectolytic enzymes, viz., polymethylgalacturonase (PMG), polygalacturonase (PG), and pectinlyase (PL) were deactivated at various combinations of pH and temperature. The deactivation process was modelled as first-order kinetics and the deactivation rate constant was found to be minimum at pH 2.2 and 23 °C, respectively for PMG, 4.8 and 28 °C, respectively for PG and 3.9 and 29 °C, respectively for PL. Thermodynamic parameters like ΔG , ΔH , ΔS , and activation energies were evaluated for crude and partially purified enzymes. It was found that entropy values are negative for all the three enzymes. The partially purified enzymes were found to be less stable than the crude enzymes. Hence, the interaction effect among the enzymes and also the effect of protein (BSA) on deactivation were studied. The addition of protein doubled the half-life times of partially purified PMG, PG I and PG II. The interaction effect of pectolytic enzymes on deactivation was found to be significant.
Keywords: Pectolytic enzymes; Deactivation kinetics; Thermodynamic parameters; Interaction effect;

Two process solutions were proposed for biochemical reactions carried out in the biphase organic solvent–water system where a membrane played the role of sustaining an interface. One of the two solutions with a passive- or active-type membrane was selected on the basis of the kinetic or diffusional regime of a particular process.Enzymatic synthesis of ZAlaPheOMe, the precursor of bitter dipeptide was discussed. On the basis of analysis of relative rates of enzymatic conversion and extraction of hydrophobic (a limiting substrate)—ZAlaOH from organic to aqueous phase, the range of the process was determined using the Hatta model. Very low values of the Hatta number were obtained which indicated that the process was carried out in the kinetic regime.Such a process solution was selected in which the enzyme was dissolved in the whole volume of the aqueous phase (passive character of the membrane). The process of ZAlaPheOMe synthesis confirmed a proper choice of the process solution; during the process the substrate concentrations in the aqueous phase were close to equilibrium concentrations. An intensive extraction of a hydrophobic product to the organic phase and high stability of the applied catalyst were observed.
Keywords: Membrane phase contactor; Biphase system; Peptide synthesis; Reaction regime;

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