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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.143, #2)

Inhibition Kinetics of Cabbage Butterfly (Pieris rapae L.) Larvae Phenoloxidase Activity by 3-Hydroxy-4-Methoxybenzaldehyde Thiosemicarbazone by Chao-Bin Xue; Wan-Chun Luo; Lin Jiang; Xian-Ye Xie; Ting Xiao; Lei Yan (pp. 101-114).

Phenoloxidase (PO) is a key enzyme in insect development, responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay in air-saturated solutions and the kinetic behavior of PO from Pieris rapae (Lepidoptera) larvae in the oxidation of l-tyrosine (a monophenol) and l-DOPA (l-3, 4-dihydroxyphenylalanine) (a diphenol) was studied. The inhibitory effects of 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone (3-H-4-MBT) on the monophenolase and diphenolase activities of PO were also studied. The results show that 3-H-4-MBT can inhibit both the monophenolase and diphenolase activities of PO. The lag period of l-tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activities of the enzyme sharply decreased. The inhibitor was found to be noncompetitively reversible with a K _I (K _I = K _IS) of 0.30 μmol/L and an estimated IC₅₀ of 0.14 ± 0.02 μmol/L for monophenolase and 0.26 ± 0.04 μmol/L for diphenolase. In the time course of the oxidation of l-DOPA catalyzed by the enzyme in the presence of different concentrations of 3-H-4-MBT, the rate decreased with increasing time until a straight line was approached. The microscopic rate constants for the reaction of 3-H-4-MBT with the enzyme were determined.

Keywords: Phenoloxidase; Inhibition kinetics; 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone; Monophenolase; Diphenolase; Pieris rapae (L.)

Evaluation of Dry Protonated Calcium Alginate Beads for Biosorption Applications and Studies of Lead Uptake by Ricardo Lagoa; J. R. Rodrigues (pp. 115-128).

Alginate polysaccharide is a promising biosorbent for metal uptake. Dry protonated calcium alginate beads for biosorption applications were prepared, briefly characterized and tested for lead uptake. Several advantages of this biosorbent are reported and discussed in comparison with other alginate-based sorbents. The alginate beads contained 4.7 mmol/g of COOH groups, which suffered hydrolysis near pH 4. The Weber and Morris model, applied to kinetic results of lead uptake, showed that intraparticle diffusion was the rate-controlling step in lead sorption by dry alginate beads. Equilibrium experiments were performed and the data were fitted with different isotherm models. The Langmuir equation was the most adequate to model lead sorption. The maximum uptake capacity (q _max) was estimated as 339 mg/g and the Langmuir constant (b) as 0.84 l/mg. These values were compared with that of other sorbents found in the literature, indicating that dry protonated calcium alginate beads are among the best biosorbents for the treatment and recovery of heavy metals from aqueous streams.

Keywords: Biosorption; Alginate; Lead; Heavy metal; Water treatment; Metal recovery; Polysaccharide

Purification and Characterization of a Cu,Zn-SOD from Garlic (Allium sativum L.). Antioxidant Effect on Tumoral Cell Lines by Imen Hadji; M. Nejib Marzouki; Daniela Ferraro; Elena Fasano; Hafedh Majdoub; Giovanni Pani; Ferid Limam (pp. 129-141).

Crude garlic extract contains one Mn-superoxide dismutase designated as SOD1 and two Cu,Zn superoxide dismutases as SOD2 and SOD3. The major isoform SOD2 was purified to homogeneity by Sephacryl S200-HR gel filtration, DEAE Sepharose ion exchange chromatography, and chromatofocusing using PBE 94. SOD2 was purified 82-fold with a specific activity of 4,960 U/mg protein. This enzyme was stable in a broad pH range from 5.0 to 10.0 and at various temperatures from 25 to 60°C. The native molecular mass of SOD2 estimated by high performance liquid chromatography on TSK gel G2000SW column was 39 kDa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a single band near 18 kDa, suggesting that native enzyme was homodimeric. The isoelectric point as determined by chromatofocusing was 5. Analysis of its N terminal amino acid sequence revealed high sequence homology with several other cytosolic Cu,Zn-SODs from plants. Exposure of cancer cell lines to garlic Cu,Zn-SOD2 led to a significant decrease in superoxide content with a concomitant rise in intracellular peroxides, indicating that the enzyme is active in mammalian cells and could, therefore, be used in pharmacological applications.

Keywords: Cu,Zn-SOD; Antioxidant activity; Garlic; Purification; Tumoral cell lines; Reactive oxygen species

Neural Network Inference of Molar Mass Distributions of Peptides during Tailor-Made Enzymatic Hydrolysis of Cheese Whey: Effects of pH and Temperature by Gilson A. Pinto; Raquel L. C. Giordano; Roberto C. Giordano (pp. 142-152).

The fine-tuning of the enzymatic hydrolysis of proteins may provide a pool of peptides with predefined molar mass distributions. However, the complex mixture of molecules (peptides and amino acids) that results after the proteolysis of cheese whey turns unfeasible the assessment of individual species. In this work, a hybrid kinetic model for the proteolysis of whey by alcalase®, multipoint-immobilized on agarose, is presented, which takes into account the influence of pH (8.0–10.4) and temperature (40–55 °C) on the activity of the enzyme. Five ranges of peptides’ molar mass have their reaction rates predicted by neural networks (NNs). The output of NNs trained for constant pH and temperatures was interpolated, instead of including these variables in the input vector of a larger NN. Thus, the model complexity was reduced. Coupled to differential mass balances, this hybrid model can be employed for the online inference of peptides’ molar mass distributions. Experimental kinetic assays were carried out using a pH-stat, in a laboratory-scale (0.03 L) batch reactor. The neural-kinetic model was integrated to a supervisory system of a bench-scale continually stirred tank reactor (0.5 L), providing accurate predictions during validation tests.

Keywords: Cheese whey proteolysis; State inference; Immobilized alcalase; Neural networks; Hybrid model; Enzymatic hydrolysis

Enzyme–Carbon Nanotube Conjugates in Room-temperature Ionic Liquids by Bilge Eker; Prashanth Asuri; Saravanababu Murugesan; Robert J. Linhardt; Jonathan S. Dordick (pp. 153-163).

Room-temperature ionic liquids (RTILs) are intriguing solvents, which are recognized as “green” alternatives to volatile organics. Although RTILs are nonvolatile and can dissolve a wide range of charged, polar, and nonpolar organic and inorganic molecules, there remain substantial challenges in their use, not the least of which is the solvents’ high viscosity that leads to potential mass transfer limitations. In the course of this work, we discovered that the simple adsorption of the bacterial protease, proteinase K, onto single-walled carbon nanotubes (SWNTs) results in intrinsically high catalytic turnover. The high surface area and the nanoscopic dimensions of SWNTs offered high enzyme loading and low mass transfer resistance. Furthermore, the enzyme–SWNT conjugates displayed enhanced thermal stability in RTILs over the native suspended enzyme counterpart and allowed facile reuse. These enzyme–SWNT conjugates may therefore provide a way to overcome key operational limitations of RTIL systems.

Keywords: Room-temperature ionic liquids; Enzyme–SWNT conjugates; Diffusional limitations; Enzyme kinetics; Thermostability; Reusability

Highly Sensitive Determination of DAO Activity by Oxidation of a Luminescence Reagent by I. Mayer; F. Pittner; M. Hermann; A. Missbichler (pp. 164-175).

A highly sensitive method for measuring the activity of the enzyme diamine oxidase (DAO) independent of the type of substrate is described. The principle of the assay is to determine the amount hydrogen peroxide generated as a reaction product during oxidation of diamines by DAO. PSatto™, a highly sensitive luminescence reagent, was used to generate a signal depending on the hydrogen peroxide concentration based on the action of horseradish peroxidase. DAO is specifically captured from a sample by an antibody immobilized to microwell plates, and the substrate is added to the bound enzyme. Various diamines were used as substrates; the peroxide produced is directly proportional to the amount of DAO bound to the specific antibodies. With this very sensitive method, it is possible to detect pmol amounts of generated hydrogen peroxide in plasma matrix corresponding to the biological activity of DAO.

Keywords: Diamine oxidase; Luminescence; Putrescine; Histamine; Antibody; Purification

Polyploid Formation Between Aspergillus niger and Trichoderma viride For Enhanced Citric Acid Production From Cellulose by Ramida Watanapokasin; Nitisak Sawasjirakij; Shoji Usami; Kohtaro Kirimura (pp. 176-186).

The first-stage heterokaryons, obtaining from intergeneric protoplast fusion between Aspergillus niger (Y-b) and Trichoderma viride (M5S51), showed slow growth and mixed morphologies on minimal medium. The fusants were classified into heterokaryon and prototrophic haploid, showing the morphology as that of A. niger. The heterokaryon strains formed conidia with the same nutritional requirements as those of the original auxotrophic mutant strains. After several subcultivations on minimal medium containing d-camphor, some heterokaryon strains formed larger two to seven nuclei/conidium as compared to one nucleus/conidium of the auxotrophic mutant and prototrophic strains, indicating that the new hybrids were generated. Interestingly, three fusant strains AT 11-2-3, AT 11-2-10, and AT 11-2-14 produce 19.2, 6.1, and 10.5 g/l citric acid, respectively, in semisolid culture containing cellulose, whereas A. niger Yang no. 2 could not use carboxymethyl cellulose as the sole carbon source for citric acid production. In addition, the average maximum β-glucosidase and carboxymethylcellulase productions from AT 11-2-3, AT 11-2-10, and AT 11-2-14 were about 16- and 4-folds higher than those of A. niger, respectively.

Keywords: Aspergillus niger ; Intergeneric fusant; Citric acid production; Polyploid; Trichoderma viride

Extraction of Proteins from Switchgrass Using Aqueous Ammonia within an Integrated Biorefinery by Bryan Bals; Laura Teachworth; Bruce Dale; Venkatesh Balan (pp. 187-198).

Switchgrass (Panicum vergatum) is a potential feedstock for future cellulosic biorefineries. Such a feedstock may also provide protein, most likely for use as an animal feed. In this paper, we present a potential scheme for integrating fiber processing with extractions to obtain both sugar and protein products from switchgrass pretreated using Ammonia Fiber Expansion (AFEX). Solutions of 3% aqueous ammonia at pH 10.5 provided optimal extraction of proteins. Addition of the nonionic surfactant Tween-80 improved protein recovery for AFEX-treated materials. It was determined that an extraction following AFEX solubilized approximately 40% of the protein, while a subsequent hydrolysis solubilized much of the remaining protein while producing 325 g sugar per kg biomass. The remaining insoluble residue contained very little protein or ash, making it ideal for heat and power production. In contrast, an extraction following hydrolysis solubilized only 68% of the original protein in the biomass, while obtaining slightly higher sugar yields.

Keywords: AFEX; Switchgrass; Extraction; Enzymatic hydrolysis; Leaf protein

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.143, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.143, #2)