Biochemical Engineering Journal (v.41, #1)

BEJ Keywords (II).

The empirical kinetic models for the resinoid extraction from St. John's worth (Hypericum perforatum L.) aerial parts were analyzed to choose the optimum one regarding their relative simplicity and accuracy of fitting the experimental data obtained at different operating conditions. The following two-parametric models were analyzed: a parabolic diffusion model, a power law equation, a hyperbola equation, an exponential equation of the Weibull type and a logarithmic relation of the Elovich type. The aqueous solutions of ethanol (70 and 95% by volume) were used to isolate the resinoid from a charge of the ground plant material (the plant material-to-solvent ratio: 1:5 and 1:10 g/mL; the extraction temperature: 25, 50 and about 80 °C; and the mean plant particle size: 0.23, 0.57 and 1.05 mm). All empirical models gave a good fit to the experimental data (root mean square, RMS < ±5%), but the best one was Elovich's equation having the smallest RMS (±2.5%) and the highest linear correlation coefficient (0.975). The best empirical model was somewhat better than the physical model based on the film theory (RMS = ±2.8%). The effects of the process factors on the kinetic model parameters were assessed using the full factorial test plan 24.
Keywords: Solid–liquid extraction; Extraction kinetics; Empirical models; St. John's worth; Hypericum perforatum L.; Full factorial plan;

By use of PCR, the genes encoding d-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coli BL21 (DE3) to overexpress d-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (PHase) whose optimal length was confirmed to 209 bp. Furthermore, the RBS region in the downstream of PHase was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E. coli strain Top10F′. The enzyme activity of Top10F′/pGEMT-R-DCB grown at 37 °C was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 °C.
Keywords: Hydantoinase promoter; d-Carbomoylase; Constitutive expression; d-p-Hydroxyphenylglycine (d-HPG);

Development of AC microelectrophoresis for rapid protein affinity evaluation by Tomoko Okada; Yuji Yamamoto; Tsubasa Shibuya; Hyen-Wook Kang; Hirotaka Miyachi; Isao Karube; Hiroshi Muramatsu; Jong Min Kim (17-23).
We have developed a new method for evaluating the affinity interactions between two different proteins by applying an alternating current (AC) voltage to a micro-flow channel. An AC voltage was applied to the protein-modified microspheres in the micro-flow channel, which resulted in the oscillation of the microspheres owing to their surface charges. The oscillation amplitude showed a linear relationship with the charge density of the microspheres. As an example for protein affinity measurement, the amplitude changes of a profilin-modified microsphere were measured by the addition of actin. In the same electrical condition, the oscillation amplitude of the profilin-modified microsphere increased by ≈175% by binding with actin. Similar results in the principle were obtained for the affinity interaction between biotin and streptavidin. The results showed that the higher the charge density of the microspheres induced by binding with different proteins, the higher the oscillation amplitude of the microspheres, thus, suggesting a possible application of the micro-flow channel and AC voltage on the protein property study, as well as on the biosensor application using the oscillation amplitude changes.
Keywords: Affinity; Bioprocess monitoring; Biosensors; Protein; Electrophoresis; AC voltage;

A downstream process strategy for a whole microbial transformation to produce l-phenylacetylcarbinol (PAC) in a nonionic surfactant Triton X mediated cloud point system was developed. By application of a Winsor I microemulsion, the product and the nonionic surfactant in the microbial transformation broth was separated successfully. Then the nonionic surfactant was recovered with a Winsor II microemulsion. In a single stage Winsor I microemulsion extraction process, the product recovery ratio 76.9% and the nonionic surfactant recovery ratio 66.5% were achieved. A discrete countercurrent extraction operation was also carried out to improve the separation efficiency. Finally, character of the recovery nonionic surfactant was also examined.
Keywords: Nonionic surfactant; Cloud point system; Liquid–liquid extraction; Biotransformations; Microemulsions; Downstream processing;

The lipase from filamentous fungi Rhizopus chinensis, as a membrane-bound enzyme, possesses the excellent catalysis ability for esterification and transesterification reactions, and has a good potential in many industrial applications. In order to improve the synthetic activity of the lipase, the effects of oils and oil-related substrates on its production and the fermentation media optimization were investigated. Based on the results, it was suggested that oleic acid could be the important substrate for the lipase production. Among various oils and oil-related substrates, olive oil containing high content of oleic acid was the optimal one for the lipase production. Using orthogonal test and response surface methodology (RSM), the composition of fermentation media was further optimized. The optimized media for lipase synthetic activity and activity yield was composed of peptone 57.94 and 55.58 g L−1, olive oil 21.94 and 22.99 g L−1, maltose 12.91 and 14.34 g L−1, respectively, with K2HPO4 3 g L−1, MgSO4·7H2O 5 g L−1 and initial pH 6.0. Under the optimal conditions, the lipase activity and the activity yield were improved 61.5 and 93.4% comparing the results before optimization, respectively. The adequate models obtained had predicted the lipase production successfully.
Keywords: Rhizopus chinensis; Lipase; Enzyme activity; Fermentation; Medium composition; Optimization;

The growth of the yeast Saccharomyces cerevisiae, the fungus Rhizopus nigricans and Nicotiana tabacum cells with perfluorodecalin as an oxygen carrier has been studied. The volumetric mass transfer coefficient (k L a) measured by the dynamic method was higher for the perfluorodecalin oxygenation system than for the conventional aeration system. The results show that perfluorocarbon can be successfully used as an efficient gas carrier, especially for the culture of delicate plant cells. The increase in yeast biomass in the suspension culture aerated by perfluorodecalin was as much as 110% higher than in the culture aerated by air. The fungus R. nigricans grew better when the conventional aeration system was used due to the fact that growth of the mycelium is limited by the transport of oxygen by diffusion in the pellets rather than by interfacial oxygen transport. In the case of isolated tobacco cells, an increase of over 350% in biomass growth was observed for the PFC aeration system.
Keywords: Perfluorocarbon; Perfluorochemical; Oxygen carrier (vector); Microbial/plant cell suspension culture;

Thermal degradation kinetics of the phycocyanin from Spirulina platensis by Francine S. Antelo; Jorge A.V. Costa; Susana J. Kalil (43-47).
The cyanobacterium Spirulina platensis is a source of pigments, such as phycocyanin, which is used in the food, cosmetic and pharmaceutical industries. The thermal degradation kinetics of the liquid extract at pH values of 5, 6 and 7 was studied, evaluating its stability between 50 and 65 °C. The kinetic model was assumed and validated as being of the first order. Between 50 and 55 °C the extract was more stable at pH 6 and between 57 and 65 °C at pH 5, but was shown to be increasingly unstable at pH 7 as the temperature of the treatment increased. The addition of sorbitol between 10 and 50% (w/w) in the treatment at 62 °C for 30 min increased the half-life values of the phycocyanin extract, proving that its de-colorization was related to degradation of the protein chain.
Keywords: Phycocyanin; Sorbitol; Protein; Microalgae; Biokinetics; Kinetic parameters;

Isolation of γ-aminobutyric acid-producing bacteria and optimization of fermentative medium by Xiaoxue Lu; Zhigang Chen; Zhenxin Gu; Yongbin Han (48-52).
Ten γ-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) strains were isolated from kimchi and yoghurt. The strain B, isolated from kimchi showed the highest GABA-producing ability (3.68 g/L) in MRS broth with 1% monosodium glutamate (MSG). Strain B was identified as Lactococcus lactis subsp. lactis. The GABA-producing ability of L. lactis B was investigated using brown rice juice, germinated soybean juice and enzymolyzed skim milk as medium compositions. The D-optimal mixture design was applied to optimize the ratio of the three kinds of components in the media. The results showed that when the mixing ratio of brown rice juice, germinated soybean juice and enzymolyzed skim milk was 33:58:9 (v:v:v), the maximum GABA yield of L. lactis B was 6.41 g/L.
Keywords: Amino acid; Lactic acid bacteria; Fermentation; D-optimal mixture design optimization; Biotransformation; Food engineering;

In this paper, a feedforward–cascade controller for dissolved oxygen concentration in an activated sludge process is designed in order to meet stricter effluent quality standards at a minimum cost. Conventional proportional and integral (PI) constant dissolved oxygen set-point control and feedforward–cascade dissolved oxygen set-point control are evaluated using the reduced model of activated sludge model no. 1 and reduced IWA simulation benchmark. The feedforward–cascade control has been based on a hierarchical structure where a high level or cascade control selects the set-point of the low level or conventional controller and low level directly control dissolved oxygen concentration. And feedforward control is introduced in the control system for preventing the influent loading from influencing the system. Simulation results show that feedforward–cascade control of the activated sludge process is more successful than conventional PI control in meeting the effluent standards and reducing operational costs. This control strategy can be expected to be accepted by the operating personnel in wastewater treatment plants.
Keywords: Activated sludge process; ASM1; Dissolved oxygen concentration; Feedforward control; Cascade control;

A bioreactor system with 30 packed gel envelopes was installed in a thermal power plant for the removal of nitrogen from ammonia-containing desulfurization wastewater. Each envelope consisted of double-sided plate gels containing Nitrosomonas europaea and Paracoccus denitrificans cells with an internal space in between for injecting an electron donor. The envelope can remove ammonia from wastewater in a single step. When the wastewater was continuously treated with the bioreactor system, it removed 95.0% of the total nitrogen in the inlet, and the total nitrogen concentration in the outlet was below 9.0 mg L−1. The maximum nitrogen removal rate was 6.0 g day−1 per square meter of the gel area. The maximum utilization efficiency of the injected ethanol for denitrification was 98.4%, and the total organic carbon concentration in the outflow was maintained at a low level. Since the bioreactor system could use the electron donor effectively, it was not necessary to use an additional aerobic tank to remove the electron donor and a settling tank to segregate the surplus sludge containing bacteria from wastewater. Our concept of using packed gel envelopes would be highly effective for constructing a simple and efficient nitrogen removal system capable of simultaneous nitrification and denitrification.
Keywords: Nitrogen removal; Wastewater treatment; Bioreactor; Immobilized bacteria; Nitrification; Denitrification;

Polyhydroxyalkanoate (PHA) synthase PhaC plays a very important role in biosynthesis of microbial polyesters PHA. Compared to the extensively analyzed C-terminus of PhaC, N-terminus of PhaC was less studied. In this paper, the N-terminus of two class I PHA synthases PhaCRe and PhaCAh from Ralstonia eutropha and Aeromonas hydrophila, respectively, and one class II synthase PhaC2Ps of Pseudomonas stutzeri strain 1317, were investigated for their effect on PHA synthesis. For PhaCRe, deletion of 2–65 amino acid residues on the N-terminus led to enhanced PHB production with high PHB molecular weight of 2.50 × 106  Da. For PhaCAh, the deletion of the N-terminal residues resulted in increasing molecular weights and widening polydispersity accompanied by a decreased PHA production. It was found that 3-hydroxybutyrate (3HB) monomer content in copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (3HHx) increased when the first 2–9 and 2–13 amino acid residues in the N-terminus of PhaC2Ps were deleted. However, deletion up to the 40th amino acid disrupted the PHA synthesis. This study confirmed that N-terminus in different types of PHA synthases showed significant roles in the PHA productivity and elongation activity. It was also indicated that N-terminal mutation was very effective for the location of functional regions at N-terminus.
Keywords: Polyhydroxyalkanoates; PHA synthase; DNA; Recombinant DNA; Biosynthesis; Amino acids;

A new fermentation strategy for S-adenosylmethionine production in recombinant Pichia pastoris by Jianguo Zhang; Xuedong Wang; Erzheng Su; Guochen Fang; Yuhong Ren; Dongzhi Wei (74-78).
A recombinant Pichia pastoris MutS expressing SAM2 gene of Saccharomyces cerevisiae was cultured for S-adenosylmethionine (SAM) accumulation. Effect of the amount of methanol added (0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 6.0%, 10.0%, and 12.0%) and cell densities (9.57, 13.47, 21.74, 30.90, and 41.24 g/L dry cell weight (DCW)) on yield of SAM was found in flask cultivations. In flask experiments, maximal yield of SAM (1.29 g/L) was obtained at 2.0% methanol added and 30.90 g/L DCW which gave the maximal methanol consumption rate. Conjunct effect of amount of methanol added and cell density was found through Origin 7.0 (7.0 Microcal, USA). Scale up in 3.7 L bioreactor, 51% specific yield of SAM was enhanced at 0.6% methanol compared to that of 0.1% methanol. In fed-batches of different cell densities at 0.6% methanol, maximal yield of SAM was 8.66 g/L at 100 g/L DCW with 64% yield of SAM enhanced again. Methanol consumption rate at 100 g/L DCW was 4.81 mL/L h. Maintenance coefficient of 100 g/L DCW was lower than that of others significantly, although methanol consumption rate of 90 g/L DCW was higher (5.07 mL/L h) than that of 100 g/L DCW.
Keywords: High cell density fermentation; Methanol consumption rate; S-adenosylmethionine; Pichia pastoris;

Cyclodextrin production by Bacillus firmus strain 37 immobilized on inorganic matrices and alginate gel by Cassiana Mazzer; Lívia Rosas Ferreira; Júlia Regina Tedesco Rodella; Cristiane Moriwaki; Graciette Matioli (79-86).
Production of β-cyclodextrin (β-CD) by Bacillus firmus strain 37 cells, immobilized by adsorption on silica–titania (SiO2/TiO2) and silica–manganese dioxide (SiO2/MnO2) matrices, was optimized for temperature, substrate concentration and initial biomass. The immobilization process was most efficient at 60 °C with 10% maltodextrin and 1.0 g of cells, resulting, after a 5-day assay, in a β-CD production of 11.7 ± 0.1 mM for cells immobilized on SiO2/TiO2 and 11.2 ± 0.1 mM in SiO2/MnO2. Entrapment in alginate gel resulted in a maximum β-CD production of 4.1 ± 0.1 mM, which was maintained constantly until the end of a 10-day assay. During this same period, free cells produced 8.3 ± 0.2 mM, and cells immobilized on SiO2/TiO2 and SiO2/MnO2, 16.7 ± 0.4 and 17.3 ± 0.5 mM, respectively. β-CD production by cells immobilized in calcium alginate in four repetitive cycles of 5 days each, showed an increase up to the third cycle, reaching 4.8 ± 0.2 mM, while production by free cells started falling from the second cycle. In this same assay, cells immobilized on SiO2/TiO2 and SiO2/MnO2, showed the best β-CD production results at the end of the first cycle, with a gradual fall occurring due to the desorption of cells thereafter.
Keywords: Bacillus firmus; Cyclodextrins; Immobilized cells; Adsorption; Batch processing; Enzymes;

The kinetics of the esterification of oleic acid with 1-butanol catalyzed by free Rhizomucor miehei lipase in a biphasic system was studied in a batch reactor. The reaction appeared to proceed via a Ping Pong bi–bi mechanism with 1-butanol inhibition. The kinetic constants of the model were determined from experiments at 30 °C with initial concentrations of 0.1 – 0.95 mol L org − 1 oleic acid and 0.1 – 1 mol L org − 1 1-butanol in the organic phase and 0.05–0.2 g L−1 enzyme in the aqueous phase. The model was used to simulate the batch concentration profiles of the product as well as the initial reaction rates. Agreement of the model with both the batch concentration profiles (average error of 7.2%) and the initial reaction rate per experiment (average error of 16.0%) was good.
Keywords: Biphasic catalysis; Esterification reaction; Rhizomucor miehei lipase; Dynamic modeling; Substrate inhibition; Kinetic parameters;

Kinetic modelling of aldolase-catalyzed addition between dihydroxyacetone phosphate and (S)-alaninal by Trinitat Suau; Gregorio Álvaro; M. Dolors Benaiges; Josep López-Santín (95-103).
This paper is focused on the development of a kinetic model for an aldolase-catalyzed reaction. The aldol addition between dihydroxyacetone phosphate (DHAP) and (S)-benzyloxycarbonyl-alaninal ((S)-Cbz-alaninal) catalyzed by the four DHAP-dependent aldolases is a promising way for the synthesis of four complementary diastereoisomers with potential biological activity. The reaction catalyzed by fuculose-1-phosphate aldolase (FucA) conducts to a synthesis product with a 100% diastereomeric excess. A kinetic model has been proposed including both the synthesis and a parallel non-desired secondary reaction. The model involved an ordered two-substrate mechanism for the synthesis and non-competitive inhibition by (S)-Cbz-alaninal and competitive inhibition by methylglyoxal byproduct in both reactions. The values of the model kinetic parameters were determined and the model validated in batch and fed-batch synthesis reactions. The obtained model could be extended to explain the behavior of other class II DHAP-dependent aldolases and exploited in simulation for reactor design purposes.
Keywords: Aldolases; Kinetic model; Stereoselective synthesis; Fuculose-1-phosphate aldolase;