Biochemical Engineering Journal (v.10, #1)

Bej keywords (IV).

Congo Red attached monosize poly(HEMA-co-MMA) microspheres for use in reversible enzyme immobilisation by Handan Yavuz; Gülay Bayramoğlu; Yasemin Kaçar; Adil Denizli; M. Yakup Arıca (1-8).
Monosize and non-porous poly(2-hydroxyethylmethacrylate-co-methylmethacrylate) (poly(HEMA-co-MMA)), microspheres were prepared by dispersion polymerisation of HEMA and MMA in an ethanol–water medium in the presence of an initiator (α,α′-azobisisobutyronitrile, AIBN). An affinity dye, i.e. Congo Red (CR) was attached covalently and then Fe3+ ions were incorporated. The poly(HEMA-co-MMA)-CR attached and poly(HEMA-co-MMA)-CR-Fe3+ incorporated microspheres were used in the immobilisation of glucose oxidase (GOD) via adsorption. The adsorption capacities of these microspheres were determined by varying the concentration of GOD in the adsorption medium. GOD adsorption capacities of the Fe3+ incorporated microspheres (165 mg g−1) was greater than that of the dye-attached microspheres (126 mg g−1). The non-specific adsorption of the GOD on the poly(HEMA-co-MMA) microspheres was negligible. The K m values for both immobilised poly(HEMA-co-MMA)-CR-GOD (7.2) and poly(HEMA-co-MMA)-CR-Fe3+-GOD (6.8) were higher than that of the free enzyme (6.6 mM). Optimum reaction pH was 5.0 for free and 7.0 for both immobilised preparations. Optimum reaction temperature of the adsorbed enzymes was 10 °C higher than that of the free enzyme and was significantly broader. After 10 successive uses the retained activity of the adsorbed enzyme was 93%. It was observed that enzyme could be repeatedly adsorbed and desorbed on the CR attached poly(HEMA-co-MMA) microspheres without significant loss in adsorption capacity or enzyme activity.
Keywords: Glucose oxidase; Immobilisation; Adsorption; Congo Red; Enzyme technology; Affinity microcarriers; Poly(HEMA-co-MMA);

An unstructured model was previously developed to describe growth of Lactobacillus helveticus; this model accounted for the biomass kinetics until the stationary state was achieved. In the new model, the decline phase was modelled as an exponential decay, all the parameters having an obvious biological meaning. By means of both expressions, the whole growth history is described. This model has been tested for a wide range of yeast extract (YE) supplementations (5–30 g l−1), i.e. for various autolysis kinetics (from 8 to 26% of autolysis after 8 h of death phase); in all cases, the new model fitted accurately the experimental data.
Keywords: Lactic acid bacteria; Growth phases; Autolysis; Model; Nitrogen supplementation; Batch cultures;

In this work, α-lactalbumin (α-la) rich precipitate particles are formed and aged in a batch stirred-tank from a whey protein concentrate (WPC) dispersion. Precipitation of the proteins occurs during a period of acid-addition followed by an ageing period. This study investigates how stirred-tank impeller agitation and subsequent processing, by means of passing precipitate suspensions through a capillary tube or a partially open ball-valve, affect particle size and composition. Precipitate particles are largely unaffected when subjected to laminar capillary tube flow. However, as flow becomes transitional and thereafter turbulent, particle breakage increases, especially for precipitates formed and aged under mild agitation conditions. Precipitates passed through the ball-valve experience even greater particle size reduction as a sharp geometrical transition results in highly turbulent flow. Moreover, particles formed and aged under low shear conditions, though initially larger, are in fact weaker and fragment to a greater extent during turbulent processing through the ball-valve. This has process design implications for separation processes where particle size is important, as shear history can influence particle toughness. Substantial size reduction of particles can best be mitigated by identifying regions of high turbulence or sudden changes in flow geometry, and by redesigning these regions so as to reduce these effects.
Keywords: Agitation; Bioprocess design; Precipitation; Protein recovery; Separation; Whey;

Stabilization of human haemoglobin by naturally occurring osmolytes by Roberto Di Domenico; Roberto Lavecchia (27-30).
The influence of the naturally occurring osmolytes xylitol, glycine and betaine on the thermal stability of human haemoglobin was investigated. Experiments were made in the temperature range of 55–70 °C, adding up to 30% w/w of osmolytes to the protein solution. All the additives stabilized haemoglobin, with xylitol and glycine appearing more effective. A kinetic analysis based on the Lumry–Eyring inactivation scheme showed that the denaturation process can be described by a second-order rate expression, with an apparent activation energy ranging from 45 to 82 kcal/mol.
Keywords: Biomedical; Haemoglobin; Kinetic parameters; Osmolytes; Protein denaturation; Sterilization;

Kinetic model for dynamic response of three-phase fluidized bed biofilm reactor for wastewater treatment by Satoshi Tsuneda; Joseph Auresenia; Yutaka Inoue; Yuji Hashimoto; Akira Hirata (31-37).
Step changes in inlet concentration has been introduced into the completely mixed three-phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the dynamic behavior of the system and to establish the suitable kinetic model from the response curve. Three identical reactors having different biomass volumes were operated in parallel. It was found that the response curves showed second-order characteristics, and thus at least two first-order differential equations are necessary to simulate the substrate and biomass response curves. Nonlinear regression analysis was performed using different types of rate equations and their corresponding kinetic parameters were used to simulate the theoretical response curve using the Runge–Kutta numerical integration method. As a result, although various types of conventional biokinetic models such as Monod, Haldane and Andrew types were examined, all the theoretical substrate response curves underestimated time constants compared to the actual substrate response plots. On the other hand, the theoretical curve of the kinetic model that incorporates adsorption term has best fit to the actual response in most of the cases. Thus, it was concluded that adsorption of substrate onto biofilm and carrier particles has significant effect on the dynamic response in biofilm processes.
Keywords: Adsorption; Biofilm reactor; Kinetic model; Nonlinear regression analysis; Step change response; Three-phase fluidized bed;

Improvement of productivity of active form of glutamate racemase in Escherichia coli by coexpression of folding accessory proteins by Jiro Kohda; Yasunori Endo; Naotake Okumura; Yoichi Kurokawa; Kazuyo Nishihara; Hideki Yanagi; Takashi Yura; Hideki Fukuda; Akihiko Kondo (39-45).
Since two classes of folding accessory proteins, molecular chaperones and foldases, prevent the misfolding of newly synthesized polypeptides in the cell, their coexpression could be expected to improve the productivity of soluble and active recombinant proteins. Escherichia coli cytoplasmic glutamate racemase (GluR), which has five cysteine thiol groups and no disulfide bond, was selected as a model enzyme and overexpressed in E. coli. The effects of coexpressing a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active GluR in E. coli were examined. A relatively large amount of active GluR produced by mild induction with 10 μM isopropyl-β-d-thiogalactopyranoside (IPTG). Active GluR productivity was further increased 2.2–2.3-fold by coexpression of GroEL/ES, Trx, or DsbB–DsbD (DsbBD), while it was decreased by coexpression of DnaK–DnaJ–GrpE and TF. These results demonstrate that coexpression of appropriate folding accessory proteins could significantly improve the productivity of active form of proteins in E. coli.
Keywords: Molecular chaperone; Foldase; Coexpression; Protein folding; Escherichia coli;

Peroxidase production by Coprinus cinereus using rotating disk contactor by Akihiko Sakurai; Syuichi Kawamoto; José F. Abarca; Mikio Sakakibara (47-53).
Peroxidase production by Coprinus cinereus was investigated using a rotating disk contactor (RDC). The effect of the specific surface area on the productivity was evaluated by batch culture. In addition, a repeated batch culture was conducted to improve the productivity. The maximum peroxidase activity by the RDC was about 1.5 times that of the shaking culture, and the production rate was about 2.0 times greater. The specific surface area in the range from 3.1 to 3.7 cm2-biofilm/cm3-medium was most suitable for the peroxidase production. In the repeated batch culture, the maximum activity slightly lowered in comparison with the batch culture, and the production rate rose by 1.7 times.
Keywords: Peroxidase; Coprinus cinereus; Rotating disk contactor; Biofilms; Immobilized cells; Enzyme production;

Arthromyces ramosus peroxidase (ARP) was successfully modified with a synthetic surfactant for one-electron oxidation reaction of a hydrophobic substrate in toluene. Although UV–visible absorption spectrum of surfactant–ARP complex in toluene showed slight red shift of Soret band compared to that in water, the complex can catalyze oxidation reaction of o-phenylenediamine (o-PDA) with hydrogen peroxide. It appeared that thermodynamic water activity in the reaction system has dominant effect on either the catalytic activity or the stability in the catalytic cycle. Steady-state kinetics under the optimal condition revealed that the specific constant (k cat/K m) of ARP complex for o-PDA was 2 orders of magnitude lower than that in aqueous media, while only 13-fold lower for hydrogen peroxide. The reduction of catalytic activity caused by altering the reaction media from water to toluene was found to be mainly due to the low specific constant of ARP complex for o-PDA rather than hydrogen peroxide.
Keywords: Biocatalysis; Coprinus cinereus peroxidase; Enzyme deactivation; Enzyme technology; Optimization; Thermodynamic water activity;

The effects of nitrogen source and initial glucose concentration were studied in submerged fermentation of higher fungus Ganoderma lucidum for simultaneous production of bioactive ganoderic acid and polysaccharide. The cells could not grow well when either yeast extract or peptone was used as the sole nitrogen source. However, a combined addition of 5 g/l of yeast extract and 5 g/l of peptone was optimal for the cell growth and metabolite production. Initial glucose concentration within 20–65 g/l also greatly affected the cell growth and product biosynthesis. The extracellular polysaccharide production was remarkably improved at a high initial glucose concentration. The highest levels of cell density (16.7 g DW/l), intracellular polysaccharide (1.19 g/l) and ganoderic acid (212.3 mg/l) were obtained at an initial glucose concentration of 50 g/l.
Keywords: Ganoderma lucidum; Ganoderic acid; Glucose and nitrogen; Higher fungi; Polysaccharide; Submerged fermentation;

Three kinds of excess sludge (ES) obtained from our laboratory and two municipal wastewater treatment plants (I and II) were disrupted by Dyno mill to explore the useful target substances for ES reuse. Activities of protease, amylase, glucosidase, lipase and dehydrogenase in the disrupted ES were compared. Protease exhibited a high activity compared with the other examined enzymes. Almost 69% of the protease activity in the original solution of disrupted ES from the municipal wastewater treatment plant II was recovered by protein precipitation using ammonium sulfate, while the recovered enzyme solution lost its activity by 32% of the original after preservation at −20 °C for 1 month. All the protease originated from the ESs of the laboratory, and municipal wastewater treatment plants enabled milk protein to be degraded into tyrosine at a yield of 103 μg-tyrosine/mg-protein, implying its potential application to enhance hydrolysis of protein in wastewater treatment.
Keywords: Enzyme activity; Excess sludge; Intracellular content; Mill disruption; Protease; Protein degradation; Reuse biotechnology;

Segmental distribution in potentials of lateral root budding and oxygen uptake of plant hairy roots by Kazuaki Ninomiya; Masahiro Kino-oka; Masahito Taya; Setsuji Tone (73-76).
The positional distributions in potential of lateral root budding and oxygen uptake rate were examined using the segments of madder and horseradish hairy roots with a length of 5.0×10−3  m obtained at different mean distances from the root tips of l=7.5×10−3–47.5×10−3  m. The average rate of lateral root budding and oxygen uptake rate of the roots with smaller l values were higher and both the rates gradually decreased with increase in l value. Positive relations were observed between the rates of lateral root budding and oxygen uptake of both the hairy roots. The relation indicated that the potential of lateral root budding was suppressed at the oxygen uptake rates of 0.15×10−5 and 0.32×10−5  mol O2/(h m) for madder and horseradish hairy roots, respectively.
Keywords: Plant cell culture; Hairy roots; Lateral root budding; Oxygen uptake rate;