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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.112, #3)
Improvement in thermal stability and substrate binding of pig kidney d-amino acid oxidase by chemical modification by Mikio Bakke; Naoki Kajiyama (pp. 123-131).
Chemical modification was evaluated to stabilize pig kidney d-amino acid oxidase (pkDAAO), which is required for analytical determination of d-amino acids. Optimization of modification conditions was performed to obtain high recovery yield and stability, and chemical modification at 30°C for 12 h with a highly concentrated enzyme solution gave dextran-conjugated pkDAAO with a 70% yield of activity. pkDAAO was stable at less than 55°C at pH 6.0, while the conjugated enzyme was stable even at 70°C. In addition, the conjugated enzyme showed decreased K m values for d-amino acids. Because of these outstanding charcteristics, this new material is expected to be available for use as a liquid assay reagent.
Keywords: Chemical modification; K m value; d-amino acid oxidase; soluble dextran; stabilization
Use of whey ultrafiltrate as a substrate for production of carotenoids by the yeast Rhodotorula rubra by Ginka Frengova; Emilina Simova; Dora Beshkova (pp. 133-141).
Carotenogenesis of the lactose-negative yeast Rhodotorula rubra GED5 was studied by cocultivation with Kluyveromyces lactis MP11 in whey ultrafiltrate (WU) (35, 50, and 70 g of lactose/L). Maximum yields of cell mass (24.3 g/L) and carotenoids (10.2 mg/L of culture fluid or 0.421 µ g/g of dry cells) were obtained by growing the microbial association in WU (50 g of lactose/L) in a fermentor with an airflow rate of 0.8 L/(L·min), agitation of 220 rpm, and temperature of 30°C. The identified carotenoid pigments—β-carotene, torulene, and torularhodin—reached maximum concentrations (133, 26.9, and 222.3 µg/g of dry cells, respectively) on d 5 for torulene and d 6 for β-carotene and torularhodin.
Keywords: Carotenogenesis; microbial association; Rhodotorula rubra ; Kluyveromyces lactis ; cocultivation; whey
Kinetics and bioenergetics of Spirulina platensis cultivation by fed-batch addition of urea as nitrogen source by Carlos E. N. Sassano; João C. M. Carvalho; Luiz A. Gioielli; Sunao Sato; Paolo Torre; Attilio Converti (pp. 143-150).
The cyanobacterium Spirulina platensis was cultivated in bench-scale miniponds on bicarbonate/carbonate solutions using urea as nitrogen source. To minimize limitation and inhibition phenomena, urea was supplied semicontinuously using exponentially increasing feeding rates. The average growth rates obtained alternately varying the total mass of urea added per unit reactor volume (275<m T<725 mg/L) and the total feeding time (9<t T<15 d) clearly evidenced nitrogen limitation for m T<500 mg/L and excess nitrogen inhibition above this threshold. The time behavior of the specific growth rate at variable urea feeding patterns allowed estimation of the time-dependent Gibbsenergy dissipation for cell growth under the actual depletion conditions of fed-batch cultivations. Comparison of the yield of growth on Gibbs energy obtained using either urea or KNO3 pointed to the preference of S. platensis for the former nitrogen source, likely owing to more favorable bioenergetic conditions.
Keywords: Spirulina platensis ; urea; fed-batch cultivation; kinetics; bioenergetics; microalgae production
Use of microfiltration as first step in recovery of protein a from fermentation broth by Anna Persson; Ann-Sofi Jönsson; Guido Zacchi (pp. 151-162).
The flux and transmission of protein A during microfiltration have been studied. We studied the performance of two commercial membranes: one made of nylon (Pall Ultipore Nylon66, 0.2 µm) and one of polyether sulfone (Pall Omega, 0.16 µm). The Nylon66 membrane had by far the best transmission of protein A although a previous study showed that bovine serum albumin (BSA), often used to characterize membranes, had much better transmission through the Omega membrane. The membrane manufacturer also states that the Omega membrane is the best membrane for this kind of application because it is a low-protein-binding membrane. The lower transmission of the Omega membrane for protein A was assumed to be owing to its smaller pores and higher charge density in combination with the larger Stokes radius for protein A. When the pH was lowered, the Nylon66 membrane still had the higher transmission. It can thus be concluded that a membrane that is found suitable for the recovery process of one protein is not always the best choice for the recovery process for other proteins even though the membrane is low protein binding.
Keywords: Protein A; microfiltration; transmission; critical flux; filter cake
Biosurfactant production by Bacillus subtilis using cassava-processing effluent by Marcia Nitschke; Glaucia Maria Pastore (pp. 163-172).
A cassava flour-processing effluent (manipueira) was evaluated as a substrate for surfactant production by two Bacillus subtilis strains. B. subtilis ATCC 21332 reduced the surface tension of the medium to 25.9 mN/m, producing a crude biosurfactant concentration of 2.2 g/L. The wild-type strain, B. subtilis LB5a, reduced the surface tension of the medium to 26.6 mN/m, giving a crude biosurfactant concentration of 3.0 g/L. A decrease in surfactant concentration observed for B. subtilis ATCC 21332 seemed to be related to an increase in protease activity. The biosurfactant produced on cassava effluent medium by B. subtilis LB5a was similar to surfactin.
Keywords: Biosurfactant; Bacillus subtilis ; cassava effluent; surfactin; protease
Discrimination among eight modified michaelis-menten kinetics models of cellulose hydrolysis with a large range of substrate/enzyme ratios by Rui M. F. Bezerra; Albino A. Dias (pp. 173-184).
The kinetics of exoglucanase (Cel7A) from Trichoderma reesei was investigated in the presence of cellobiose and 24 different enzyme/Avicel ratios for 47 h, in order to establish which of the eight available kinetic models best explained the factors involved. The heterogeneous catalysis was studied and the kinetic parameters were estimated employing integrated forms of Michaelis-Menten equations through the use of nonlinear least squares. It was found that cellulose hydrolysis follows a model that takes into account competitive inhibition by cellobiose (final product) with the following parameters: K m=3.8 mM, K ic=0.041 mM, k cat=2 h−1 (5.6×10−4 s−1). Other models, such as mixed type inhibition and those incorporating improvements concerning inhibition by substrate and parabolic inhibition, increased the modulation performance very slightly. The results support the hypothesis that nonproductive enzyme substrate complexes, parabolic inhibition, and enzyme inactivation (Selwyn test) are not the principal constraints in enzymatic cellulose hydrolysis. Under our conditions, the increment in hydrolysis was not significant for substrate/enzyme ratios <6.5.
Keywords: Cellulase kinetics; cellobiose inhibition; exoglucanase Cel7A; integrated Michaelis-Menten equations; Trichoderma reesei ; enzyme substrate complex