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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.127, #2)

Application of design-of-experiments procedures to optimize efficiently pretreatment of lipase for use in a nonaqueous reaction by Rafael A. Garcia; Mark R. Riley (pp. 69-78).

A variety of different pretreatments can improve the performance of enzymes in nonpolar reaction media. These pretreatments have primarily been studied in isolation; however, interactions between some pairs of pretreatments are known to exist. The presence of these interactions complicates the design of an optimum multifactor pretreatment. Modern design-of-experiments techniques allow the simultaneous optimization of two or more variables. To improve the performance of lipase in a model reaction, we used a technique called the method of steepest ascent to optimize three variables simultaneously: pretreatment pH and sodium phosphate concentration, and the concentration of acetic acid (one of the reactants) in the reaction mixture. In only 26 experimental runs, this optimization process determined a combination of variable settings that yielded a reaction product approx 180 times faster than achieved with untreated enzyme. Evidence is presented to demonstrate that locating this optimum with single-factor experiments would be inefficient. This article demonstrates the efficiency of the method of steepest ascent particularly for evaluation of enzymatic reaction conditions exhibiting significant interactions.

Keywords: Nonaqueous enzymology; lipase; pretreatment; optimization; lyophilization; esterification; fragrance

Conversion of municipal solid waste into carboxylic acids by anaerobic countercurrent fermentation by Cateryna Aiello-Mazzarri; Guillermo Coward-Kelly; Frank K. Agbogbo; Mark T. Holtzapple (pp. 79-93).

Municipal solid waste (MSW) and sewage sludge (SS) were combined and anaerobically converted into carboxylate salts by using a mixed culture of acid-forming microorganisms. MSW is an energy source and SS is a source of nutrients. In this study, MSW and SS were combined, so they complemented each other. Four fermentors were arranged in series for a countercurrent fermentation process. In this process, the solids and liquid were transferred in opposite directions, with the addition of fresh biomass to fermentor 1 and fresh liquid media to fermentor 4. An intermediate lime treatment of solids exiting fermentor 3 before entering fermentor 4 was applied to improve the product acid concentration from the untreated MSW/SS fermentations. All fermentations were performed under anaerobic conditions at 40°C. Calcium carbonate was added to neutralize the carboxylic acids and to control the pH. Iodoform was used as a methanogen inhibitor. Carboxylic acid concentration and gas composition were determined by gas chromatography. Substrate conversion was measured by volatile solids loss, and carboxylic acid productivity was calculated as the function of the total carboxylic acids produced, the amount of liquid in all fermentors, and time. The addition of intermediate lime treatment increased product concentration and conversion by approx 30 and 15%, respectively. The highest carboxylic acid concentrations for untreated MSW/SS fermentations with and without intermediate lime treatment were 22.2 and 17.7 g of carboxylic acid/L of liquid, respectively. These results confirm that adding a treatment step between fermentor 3 and fermentor 4 will increase the digestibility and acid productivity of the fermentation.

Keywords: MixAlco process; municipal solid waste; sewage sludge; fermentation; carboxylic acids

Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus by Gilian Duncan; Chris A. Tarling; Wade H. Bingle; John F. Nomellini; Mat Yamage; Irene R. Dorocicz; Stephen G. Withers; John Smit (pp. 95-110).

Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the β-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-β-d-cellobioside with a K M similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the k cat value was significantly reduced (0.08 s⁻¹ for Cex/RsaA vs 15.8 s⁻¹ for Cex) cat, likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a high yield (76 mg/L of dry wt for Cex/RsaA), but it can also be efficiently recycled. The Caulobacter S-layer secretion system therefore offers an attractive new model system for the production of particulate biocatalysts.

Keywords: Caulobacter surface (S)-layer protein RsaA; type I secretion; cellulomonas β-1,4-glycanase; cellulase; Cex; cellobiohydrolase; crosslinked enzyme crystals; particulate enzymes; immobilized biocatalysts

Effects of aeration of growth and on production of bacteriocins and other metabolites in cultures of eight strains of lactic acid bacteria by J. A. Vázquez; J. Mirón; M. P. González; M. A. Murado (pp. 111-124).

In general, it is accepted that the production of bacteriocins in lactic acid bacteria cultures implies moderately to highly restrictive conditions regarding the availability of oxygen. However, the situation appears to be more complex, probably owing to the facultative anaerobic character of these microorganisms. By studying the culture of eight strains of lactic acid bacteria carried out in vessels with different loads of medium within an interval that determines linearly the minimum availability of oxygen, the existence of three types of behavior was highlighted: production increases (1) with the availability of oxygen, (2) with the restriction of this availability, and (3) toward both extremes of these conditions, diminishing in intermediate situations. These behaviors affected not only the production of bacteriocins, but also their metabolic character (in the Luedeking and Piret sense), as well as the production of other characteristic metabolites, such as lactic acid, acetic acid, and ethanol.

Keywords: Aeration; bacteriocin production; lactic acid bacteria; aerobiosis; metabolites

Comparison of bacterial and phage display peptide libraries in search of target-binding motif by Mojca Lunder; Tomaž Bratkovič; Bojan Doljak; Samo Kreft; Uroš Urleb; Borut Štrukelj; Nadja Plazar (pp. 125-131).

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-throughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better.

Keywords: Phage display library; bacterial display library; affinity selection; ligand; peptide; streptavidin

Protection of mitochondrial integrity from oxidative stress by selenium-containing glutathione transferase by Huijun Yu; Junqui Liu; Jing Li; Tianzhu Zang; Guimin Luo; Jiacong Shen (pp. 133-142).

The antioxidant activity of a novel artificial glutathione peroxidase-like enzyme, selenium-containing glutathione 5-transferase from Lucilia cuprina (seleno-LuGST1-1), was studied by using a ferrous sulfate/ascorbate-induced mitochondrial damage model system. Swelling of mitochondria, lipid peroxidation, and cytochrome-c oxidase activity were selected to evaluate the preservation of mitochondrial integrity in this system. Seleno-LuGST1-1 could effectively protect the mitochondria against oxidative damage in a dose-dependent manner and exhibited both higher catalytic activity and greater antioxidant ability than the classic mimic, 2-phenyl-1,2-benziososelenazol-3(2H)-one (Ebselen). This novel artificial biocatalyst therefore may have great protential for pharmacologic application in the treatment of reactive oxygen species-related diseases.

Keywords: Antioxidant activity; artificial enzymes; mitochondria; oxidative stress; glutathione peroxidase; selenium-containing glutathione transferase

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.127, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.127, #2)