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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.81, #3)

Further studies on the bioconversion of penicillin G into deacetoxycephalosporin G by resting cells of Streptomyces clavuligerus NP-1 by Marco A. Báez-Vásquez; José L. Adrio; Jacqueline M. Piret; Arnold L. Demain (pp. 145-152).

Resting cells of Streptomyces clavuligerus NP-1, which posses deacetoxy-cephalosporin C synthase activity, have been shown previously to perform oxidative ring expansion of penicillin G in the presence of iron, ascorbic acid, and α-ketoglutaric acid to form deacetoxycephalosporin G. Further studies on this bioconversion indicated that use of MOPS or HEPES buffer at pH 6.5 more than doubled the extent of the reaction observed with the previously used Tris-HCl at pH 7.4. Levels of bioconversion as high as 16.5% were achieved at low penicillin G concentrations. Previously, conversion yields were <1%.

Keywords: Streptomyces clavuligerus ; β-lactam antibiotics; bioconversion; penicillin G; deacetoxycephalosporin G; resting cells

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems by Haiyin Wang; Richard W. Jones (pp. 153-160).

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.

Keywords: Cellulase; heterologous expression; Escherichia coli ; Saccharomyces cerevisiae

Analyte-receptor binding kinetics for different types of biosensors by Anand Ramakrishnan; Ajit Sadana (pp. 161-179).

A fractal analysis is presented for analyte-receptor binding kinetics for different types of biosensor applications. Data taken from the literature may be modeled using a single-fractal analysis, a single- and a dual-fractal analysis, or a dual-fractal analysis. The latter two methods represent a change in the binding mechanism as the reaction progresses on the surface. Predictive relationships developed for the binding rate coefficient as a function of the analyte concentration are of particular value since they provide a means by which the binding rate coefficients may be manipulated. Relationships are presented for the binding rate coefficients as a function of the fractal dimension D f or the degree of heterogeneity that exists on the surface. When analyte-receptor binding is involved, an increase in the heterogeneity on the surface (increase in D f) leads to an increase in the binding rate coefficient. It is suggested that an increase in the degree of heterogeneity on the surface leads to an increase in the turbulence on the surface owing to the irregularities on the surface. This turbulence promotes mixing, minimizes diffusional limitations, and leads subsequently to an increase in the binding rate coefficient. The binding rate coefficient is rather sensitive to the degree of heterogeneity, D f, that exists on the biosensor surface. For example, the order of dependence on D f1 is 7.25 for the binding rate coefficient k ₁ for the binding of a Fab fragment of an antiparaquat monoclonal antibody in solution to an antigen in the form of a paraquat analog immobilized on a sensor surface. The predictive relationships presented for the binding rate coefficient and the fractal dimension as a function of the analyte concentration in solution provide further physical insights into the binding reactions on the surface, and should assist in enhancing biosensor performance. In general, the technique is applicable to other reactions occurring on different types of surfaces, such as cell-surface reactions.

Keywords: Analyte-receptor binding; fractals; biosensors

Preparation and preliminary characterization of exopolysaccharides by yeast Rhodotorula acheniorum MC by Drinka Grigorova; Konstantza Pavlova; Ivan Panchev (pp. 181-191).

The effects of various carbon and nitrogen sources on the synthesis of exopolysaccharides by Rhodotorula acheniorum MC were studied. The dynamic viscosity of cell-free culture broths during exopolysaccharide synthesis were measured. The highest values for the viscosity (10.14 MPa·s) and crude polysaccharide productivity (6.6 g/L) were obtained in a medium supplied with 5% sucrose. Ammonium sulfate was the most favorable nitrogen source for expolysaccharide synthesis. The value of pH played a determinant role, and the obligatory condition for exopolysaccharide production was low (pH 1.7–2.0) during the fermentation. The chemical composition and sugar constituents of the crude exopolysaccharides were determined. Mannose was the main monosaccharide component, and its concentration was the highest (69.13%) in the crude expolysaccharide synthesize in the medium that included 5% sucrose as a carbon source.

Keywords: Polysaccharide; Rhodotorula acheniorum ; mannose

Effects of acetate on the growth and fermentation performance of Escherichia coli KO11 by Caroline M. Takahashi; Debora F. Takahashi; Maria L. Carvalhal; Flavio Alterthum (pp. 193-203).

Escherichia coli KO11, in which the genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) encoding the ethanolpathway from Zymomonas mobili were inserted into the chromosome, has been shown to metabolize all major sugars that are consituents of hemicellulosic hydrolysates to ethanol, in anaerobic conditions. However, the growth and fermentation performance of this recombinant bacteria may be affected by acetic acid a potential inhibitor present in hemicellulose hydrolysates in a range of 2.0–15.0 g/L. It was observed that acetate affected the growth of E. coli KO11, prolonging the lag phase and inducing loss of biomass production and reduction of growth rate. At lower pH levels, the sensitivity to acetic acid was enhanced owing to the increased concentration of the protonated species. On the other hand, the recombinant bacteria showed a high tolerance to acetic acid regarding fermentative performance. In Luria broth medium with glucose or xylose as a single sugar source, it was observed that neither yield nor productivity was affected by the addition of acetate in a range of 2.0–12.0 g/L, suggesting some uncoupling of the growth vs ethanol production.

Keywords: Escherichia coli ; recombinant; ethanol; acetate; toxicity; biomass; fermentation

Lipase immobilized on poly(VP-co-HEMA) hydrogel for esterification reaction by Mahiran Basri; Sunana Samsudin; Mansor Bin Ahmad; Che Nyonya Abdul Razak; Abu Bakar Salleh (pp. 205-217).

Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate)(poly[VP-co-HEMA]) hydrogel, and divinylbenzene was the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases was also determined. Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90∶10 exhibited the highest activity. Lipase immobilized on VP(%):HEMA(%) 50∶50 showed the highest thermal, solvent, storage, and operational stability compared to lipase immobilized on other compositions of hydrogels as well as the native lipase.

Keywords: Lipase; immobilization; hydrogel; esterification; stability

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.81, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.81, #3)