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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.94, #2)

Proteolysis of mesophilic and thermophilic α-amylases by Khosro Khajeh; Sadighe Khezre-Barati; Mohsen Nemat-Gorgani (pp. 97-109).

A comparative study was performed on limited and extensive proteolysis of mesophilic (from Bacillus amyloliquefaciens [BAA]) and thermophilic (from Bacillus licheniformis [BLA]) α-amylases using trypsin. As expected, the thermophilic enzyme showed greater resistance to digestion by the protease. While the catalytic potential of BLA was enhanced on proteolysis, that of BAA was diminished owing to this process. Combined with greater catalytic activity, a lower thermal stability was observed for BLA on proteolytic treatment. For both enzymes, the extent of proteolytic cleavage was reduced in the presence of various stabilizing agents. The digestion patterns are explained in terms of available information in the literature on the structure of these proteins, especially in relation to segmental mobility.

Keywords: Mesophilic-thermophilic α-amylases; proteolytic digestion; stabilizers; segmental mobility; apparent molar volume

Laboratory method for high-solids countercurrent fermentations by M. Kyle Ross; Mark T. Holtzapple (pp. 111-126).

Equipment and procedures were developed to study the conversion of lignocellulosic biomass to carboxylic acids using high-solids countercurrent fermentations. Countercurrent fermentations of cattle manure yielded a rapid fermentation (maximum 2.98 g of total acid/[L·d]) with high acid concentrations (maximum of 32.5 g of total acid/L), but the acid yield tended to be low (maximum of 0.24 g of total acid/g of volatile solids). Countercurrent fermentations of a mixture of 80% municipal solid waste/20% sewage sludge fermented more slowly (maximum of 1.98 g of total acid /[L·d]) with a lower acid concentration (maximum of 26.5 g of total acids/L), but higher acid yields were achieved (maximum of 0.34 g of total acid/g of volatile solids).

Keywords: Fermentation; countercurrent; carboxylic acids; volatile fatty acids; MixAlco process; high solids

Single-step purification and immobilization of penicillin acylase using hydrophobic ligands by Harshvardhan V. Adikane; Rajesh K. Singh; Dnyaneshwar M. Thakar; Sanjay N. Nene (pp. 127-134).

Five differenthydrophobic ligands immobilized on 4% (4XL) and 6% (6XL) crosslinked agarose were used to study the single-step purification of penicillin acylase from cell lysate. The 4XL gels showed relatively higher specific activity and recovery than the 6XL gels. In single-step purification, highly active enzyme (42 U/mg) was obtained using moderately hydrophobic ligand (octyl). The crude enzyme immobilized on octyl gel by adsorption showed significant operational stability over a period of 30 d at room temperature. Reactor studies demonstrated the feasibility of hydrophobic ligands as a medium for immobilization.

Keywords: Penicillin acylase; 6-aminopenicillanic acid; hydrophobic ligand; purification; immobilization; conversion

Fiber fractions from processing of barley in production and conservation of a biologic control agent by Tapani Tuomi; Mari Heino; Heikki Rosenqvist; Katrina Nordström; Simo Laakso (pp. 135-145).

Carriers are frequently used to overcome problems associated with microbial survival in soil after inoculation. Moreover, the use of carriers can prolong the shelf lives and lessen dusting of both biofungicides and biologic fertilizers. This study investigated the suitability of barley-based fiber fractions as growth media and immobilization matrices in the cultivation of a Streptomyces griseoviridis biologic control agent, as well as for the conservation of obtained biomass in dehydrated hydrogel capsules. The second main ingredient in all the examined carrier matrices was alginate. The aim was to find a hydrogel formulation suited for a production process in which all individual steps, including cultivation of the organism; downstream processing; and formulation, storage, and application of the product (i.e., biologic control agent), are carried out in the hydrogel matrix. Of the tested fractions, brewer’s spent grain was the best choice, when considering the price vs the nutrient contents as well as the storage time and ease of processing of the crude and the finished products. It seems that cereal fibers can be replenished with cereal fractions less rich in fiber but having a higher content of utilizable nutrients and, hence, better suited for the production of biomass. A high content of water-insoluble fiber favorably influenced the appearance as well as the applicability of the products.

Keywords: Biofungicide; Streptomyces sp.; hydrocolloids; alginate capsules

Modeling of saccharide utilization in primary beer fermentation with yeasts immobilized in calcium alginate by Daniela Šmogrovičová; Zoltán Dömény; Juraj Švitel (pp. 147-158).

Immobilized beer fermentation was studied using an industrial bottom-fermenting yeast strain Saccharomyces cerevisiae. The yeast cells were immobilized in 2.5% calcium alginate gel and used for brewing in a five-vessel cascade reactor. The fermentation was performed at 15°C at various flow rates. A nonstructured mathematical model was developed to simulate the performance of continuous primary fermentation of lager beer. The model was based on the following variables: maltose, maltotriose, glucose, fructose, ethanol, and cell concentration. Experimental values of these variables were determined in samples taken at regular intervals. For experimental data fitting a nonlinear regression was used. Substrate consumption was characterized by specific substrate consumption rate and saturation constant. The values of these two parameters were optimized for all four substrates. Inhibition effects of substrates and product were analyzed using various inhibition patterns. Only the inhibition effect of maltose on maltose consumption was clearly identified. A good-fitting relationship for maltose inhibition was found, and inhibition constants were calculated.

Keywords: Beer fermentation; brewing; saccharide utilization; modeling; immobilization; calcium alginate

Application of factorial design to the study of xylitol production from eucalyptus hemicellulosic hydrolysate by Eliana V. Canettieri; João B. Almeida E. Silva; Maria G. A. Felipe (pp. 159-168).

This study deals with the bioconversion of xylose into xylitol by Candida guilliermondii FTI 20037 using eucalyptus hemicellulosic hydrolysate obtained by acid hydrolysis. The influence of various parameters (ammonium sulfate, rice bran, pH, and xylose concentration) on the production of xylitol was evaluated. The experiments were based on multivariate statistical concepts, with the application of factorial design techniques to identify the most important variables in the process. The levels of these variables were quantified by the response surface methodology, which permitted the establishment of a significant mathematical model with a coefficient determination of R ²=0.92. The best results (xylitol=10.0 g/L, yield factor=0.2 g/g, and productivity=0.1 g/[L·h]) were attained with hydrolysate containing ammonium sulfate (1.1 g/L), rice bran (5.0 g/L), and xylose (initial concentration of 60.0 g/L), after 72 h of fermentation. The pH of fermentation was adjusted to 8.0 and the inoculum level utilized was 3 g/L.

Keywords: Factorial design; eucalyptus hemicellulosic hydrolysate; Candida guilliermondii ; xylitol

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from cottonseed oil and valeric acid in batch culture of Ralstonia sp. strain JC-64 by Cunjiang Song; Lixing Zhao; Shin Ono; Choichiro Shimasaki; Masami Inoue (pp. 169-178).

A Ralstonia sp. strain JC-64 that is capable of accumulating poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[3HB-co-3HV]) from cottonseed oil and valeric acid was isolated. By using a high limiting-nitrogen (HLN) mineral medium as the medium for the second stage of the fermentation process and by adding the two carbon sources at different times, a range of copolymers with 12–62 mol% of 3HV were produced from a series of HLN mineral mediums containing different compositions of cottonseed oil and valeric acid by Ralstonia sp. JC-64. The melting temperature (T m) of polyhydroxybutyrate from cottonseed oil was 174°C and that of P(3HB-co-3HV) with the highest 3HV-mol fraction (62%) was 81°C.

Keywords: Cottonseed oil; high limiting-nitrogen mineral medium; polyhydroxybutyrate; poly(3-hydroxybutyrate-co-3-hydroxyvalerate); Ralstonia sp. JC-64

Application of near-infrared spectra on temperature-controlled protein crystallization by Shih-Yao B. Hu; John M. Wiencek; Mark A. Arnold (pp. 179-196).

Large, high-quality protein crystals are required for the structural determination of proteins via X-ray diffraction. In this article, we propose a technique to facilitate the production of such crystals and validate its feasibility through simulations. An analytical method for protein aqueous solution based on a Fourier transform infrared (FTIR) spectroscopy is combined with a temperature control strategy to manipulate the extent of supersaturation during crystal growth, thus impacting crystal quality. The technique requires minimal knowledge about the growth kinetics a priori. The simulations show that, under ideal conditions, the design can be as effective as predesigned temperature programs for crystallization based on known growth kinetics. Two kinds of errors might be encountered with this design. Error in the estimated number of seed crystals can result in a growth rate deviating from the desired one. Nevertheless, the deviation is usually tolerable and system instability is unlikely to occur. Based on the standard error of our FTIR method, errors in concentration measurement are simulated. Measurement error can result in system instability and impair the control algorithm. Such errors may be compensated by limiting the temperature change taken by the control action, or by improving the measurement precision through the use of regressed concentrations. Through simulations, it is shown that the proposed design is practical and represents a significant improvement over the commonly used isothermal crystallization technique.

Keywords: Protein crystallization; simulation; Fourier transform infrared; lysozyme

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.94, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.94, #2)