Alginate gel biochip for real-time monitoring of intracellular processes in bacterial and yeast cells

A method was developed for producing cell biochips on the basis of calcium alginate. Cell immobilization in microvolumes of nontoxic alginate gel under mild conditions extended the range of testable micro-organisms. The possibility of studying the intracellular processes with alginate gel biochips was demonstrated in model experiments with Escherichia coli, Bordetella bronchiseptica, and Saccharomyces cerevisiae. Cell biochips proved to be suitable for simultaneous monitoring of nucleic acid and protein syntheses with two fluorescent dyes. The effect of chloramphenicol on nucleic acid synthesis was studied with five bacterial strains. Inducible synthesis of the green fluorescence protein (EGFP) in E. coli cells was monitored with the use of biochips. The level of EGFP synthesis correlated with the inductor concentration in the medium.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 96–102.Original Russian Text Copyright © 2005 by Fesenko, Nasedkina, Chudinov, Prokopenko, Yurasov, Zasedatelev.     

Engineering biocatalytic systems in organic media with low water content

The use of organic media in biocatalysis stems from the fact that in many cases biocatalytic processes can hardly be conducted (if at all) in aqueous solutions because of extremely low solubilities of substrates and/or unfavorable shift of the reaction equilibrium in water. The growing interest in this biotechnological area that has sprung up over the past few years has resulted in various approaches to enzyme stabilization against organic solvents. Thus, the main goal of the present review is to formulate a comprehensive classification of numerous successful nonaqueous biocatalytic systems based on a few fundamental principles. Typical examples are considered, along with the advantages and drawbacks inherent in each of the approaches discussed.     

Synthesis of -Dopa by Escherichia intermedia cells immobilized in a carrageenan gel

Escherichia intermedia cells were immobilized by entrapment in a carrageenan gel and used for -DOPA synthesis from catechol, pyruvate, and ammonia. A preparation containing 75 mg of cell per gram of gel retained 60–65% of its original activity. The effect of substrate concentrations on the initial rate of -DOPA synthesis was very similar for free and immobilized cells, and substrate inhibition was observed for the three substrates. In batch reactors, up to 7.8 g l⁻¹ of -DOPA was obtained in 20 h (productivity 0.39 g l⁻¹ h⁻¹). Cells immobilized in a carrageenan gel showed higher -DOPA synthesis, in both initial rates conditions and batch reactors, than cells immobilized in a polyacrylamide gel.     

Biotechnological applications of microbial bioconversions

Modern research has focused on the microbial transformation of a huge variety of organic compounds to obtain compounds of therapeutic and/or industrial interest. Microbial transformation is a useful tool for producing new compounds, as a consequence of the variety of reactions for natural products. This article describes the production of many important compounds by biotransformation. Emphasis is placed on reporting the metabolites that may be of special interest to the pharmaceutical and biotechnological industries, as well as the practical aspects of this work in the field of microbial transformations.     

Potential Applications of the Oxidoreductive Enzymes in the Decolorization and Detoxification of Textile and Other Synthetic Dyes from Polluted Water: A Review

ABSTRACT

Recently, the enzymatic approach has attracted much interest in the decolorization/degradation of textile and other industrially important dyes from wastewater as an alternative strategy to conventional chemical, physical and biological treatments, which pose serious limitations. Enzymatic treatment is very useful due to the action of enzymes on pollutants even when they are present in very dilute solutions and recalcitrant to the action of various microbes participating in the degradation of dyes. The potential of the enzymes (peroxidases, manganese peroxidases, lignin peroxidases, laccases, microperoxidase-11, polyphenol oxidases, and azoreductases) has been exploited in the decolorization and degradation of dyes. Some of the recalcitrant dyes were not degraded/decolorized in the presence of such enzymes. The addition of certain redox mediators enhanced the range of substrates and efficiency of degradation of the recalcitrant compounds. Several redox mediators have been reported in the literature, but very few of them are frequently used (e.g., 1-hydroxybenzotriazole, veratryl alcohol, violuric acid, 2-methoxy-phenothiazone). Soluble enzymes cannot be exploited at the large scale due to limitations such as stability and reusability. Therefore, the use of immobilized enzymes has significant advantages over soluble enzymes. In the near future, technology based on the enzymatic treatment of dyes present in the industrial effluents/wastewater will play a vital role. Treatment of wastewater on a large scale will also be possible by using reactors containing immobilized enzymes.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom‐up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead–based IM (IM‐N or IM‐C) or FT, it is observed that IM‐N with the nearly neutral solid matrix, IM‐C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM‐N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM‐N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high‐throughput bottom‐up proteomics workflow comprising IM‐N‐based rapid protein cleavage and fast CZE‐MS/MS enables the completion of protein sample preparation, CZE‐MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.     

Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.